Multiplex capture of gene and protein expression from a biological sample

ABSTRACT

Provided herein are methods, compositions, and kits for preparing biological samples for multiplex spatial gene expression and proteomic analysis, such as determining a location of a nucleic acid analyte and a protein analyte in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/867,223, filed Jul. 18, 2022, which is a continuation ofInternational Application PCT/US2022/020985, with an internationalfiling date of Mar. 18, 2022, which claims priority to U.S. ProvisionalPatent Application No. 63/162,870, filed on Mar. 18, 2021, U.S.Provisional Patent Application No. 63/214,058, filed on Jun. 23, 2021,U.S. Provisional Patent Application No. 63/245,697, filed on Sep. 17,2021, U.S. Provisional Patent Application No. 63/252,335, filed on Oct.5, 2021, U.S. Provisional Patent Application No. 63/270,230, filed onOct. 21, 2021, and U.S. Provisional Patent Application No. 63/311,703,filed on Feb. 18, 2022. The contents of each of these applications isincorporated herein by reference in their entireties.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named 47706-0307002.xml. The XML file,created on Jul. 6, 2023, is 15000 bytes in size. The material in the XMLfile is hereby incorporated by reference in its entirety.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the context of anintact tissue or a portion of a tissue, or provides substantial analytedata for dissociated tissue (i.e., single cells), but fail to provideinformation regarding the position of the single cell in a parentbiological sample (e.g., tissue sample).

Moreover, multiplex detection of different analytes (e.g., nucleic acid,protein, etc.) in the same biological sample, while preserving spatialinformation remains a challenge in the field. Current methods includetranscriptome wide spatial detection or various protein detectionmethods, however, methods that accomplish both within the spatialcontext of a biological sample are still needed.

SUMMARY

Multiplex detection of different analytes (e.g., nucleic acid, protein,etc.) in the same biological sample, while preserving spatialinformation remains a challenge in the field. As described above,current methods include transcriptome wide spatial detection or variousprotein detection methods, including immunofluorescence, however,methods that detect both spatial gene expression and spatial proteinexpression within the spatial context of a biological samplesimultaneously are still needed.

Provided herein are methods for determining the spatial location of anucleic acid and a protein from a biological sample including: a)providing a spatial array including a first and second plurality ofcapture probes where each plurality includes a spatial barcode and acapture domain, b) contacting the spatial array with a biologicalsample, c) contacting the biological sample with (i) a plurality ofanalyte capture agents, where an analyte capture agent includes ananalyte binding moiety and an oligonucleotide including an analytebinding moiety barcode and an analyte capture sequence, where theanalyte capture sequence includes a sequence complementary to a secondplurality of capture domains, and (ii) a plurality of templated ligationprobes, where one of the templated ligation probes includes a sequencecomplementary a first plurality of capture domains, d) binding theanalyte binding moiety of the analyte capture agent to a target protein,e) hybridizing the templated ligation probes to a target nucleic acidand ligating the probes to produce ligation products, f) hybridizing theligation products to the first plurality of capture domains and theanalyte capture sequences of the bound analyte capture agents to thesecond plurality of capture domains on the spatial array, and g)determining the sequence or a portion thereof of a captured ligationproduct, or a complement thereof, and the sequence of the spatialbarcode of the capture probe, or a complement thereof, that isassociated with the ligation product, and the sequence of the analytebinding moiety barcode, or a complement thereof, of the bound analytecapture agent, thereby determining the spatial location of a nucleicacid and the protein from the biological sample.

In some embodiments, the capture domains of the first plurality ofcapture probes are defined non-homopolymeric capture sequences orhomopolymeric sequences. In some embodiments, the capture domains of thesecond plurality of capture probes are defined non-homopolymericsequences or homopolymeric capture sequences. In some embodiments,capture domains of the first plurality of capture probes are differentfrom the capture domains of the second plurality of capture probes. Insome embodiments, homopolymeric sequence includes a polyT sequence andthe non-homopolymeric sequence includes a fixed sequence or a degeneratesequence. In some embodiments, the fixed sequence includes at least onesequence selected from SEQ ID NO: 1 through SEQ ID NO: 11.

In some embodiments, the nucleic acid is a RNA or DNA. In someembodiments, the RNA is a mRNA.

In some embodiments, the biological sample is a tissue sample. In someembodiments, tissue sample is a fresh-frozen tissue sample or a fixedtissue sample, where the fixed tissue sample is a formalin-fixed tissuesample, an acetone fixed tissue sample, a paraformaldehyde tissuesample, or a methanol fixed tissue sample. In some embodiments, thebiological sample is a tissue section, where the tissue section is afresh-frozen tissue section or a fixed section, and optionally, wherethe fixed tissue section is a formalin-fixed paraffin-embedded tissuesection, an acetone fixed tissue section, a paraformaldehyde tissuesection, or a methanol fixed tissue section. In some embodiments, thetissue sample is derived from a biopsy sample or a whole rodent embryo.

In some embodiments, before step (b) the biological sample isdeparaffinized and decrosslinked. In some embodiments, thedecrosslinking includes the use of a buffer. In some embodiments, thebuffer includes Tris-EDTA buffer at a pH from about 8 to about 10 and atemperature from about 60° C. to about 80° C. In some embodiments, thebuffer includes citrate buffer at a pH from about 5 to about 7 and atemperature from about 70° C. to about 100° C.

In some embodiments, the hybridizing the templated ligation products andthe analyte capture sequences of the bound analyte capture agentsincludes permeabilizing the biological sample.

In some embodiments, the analyte capture sequence of the oligonucleotideis blocked prior to binding to the target protein. In some embodiments,the oligonucleotide of the analyte capture agent is blocked by ablocking probe. In some embodiments, the blocking probe is removed priorto hybridizing the analyte capture sequence of the oligonucleotide ofthe analyte capture sequence to the capture domain of the capture probe.

In some embodiments, the determining in step (g) includes: a) extendingthe captured ligation products and the captured oligonucleotides of theanalyte capture agents, where the extension products include the spatialbarcode or a complement thereof, b) releasing the extension products, orcomplements thereof, from the spatial array, c) producing a library fromthe released extension products or complements thereof, and d)sequencing the library.

In some embodiments, prior to step (c) the method includespre-amplifying the extension products, or complements thereof. In someembodiments, the complement of the oligonucleotide of the analytecapture agent includes an analyte binding moiety barcode specific to theanalyte binding moiety of the analyte capture agent.

In some embodiments, the first and second plurality of capture probesinclude a cleavage domain, one or more functional domains, a uniquemolecular identifier, and combinations thereof.

In some embodiments, the method includes imaging the biological sample.In some embodiments, the imaging includes one or more of expansionmicroscopy, bright field microscopy, dark field microscopy, phasecontrast microscopy, electron microscopy, fluorescence microscopy,reflection microscopy, interference microscopy and confocal microscopy.

In some embodiments, the method includes staining the biological sample.In some embodiments, the staining includes hematoxylin and eosin. Insome embodiments, the staining includes the use of a detectable labelselected from the group consisting of a radioisotope, a fluorophore, achemiluminescent compound, a bioluminescent compound, or a combinationthereof.

In some embodiments, the spatial array includes one or more proteindilution series.

Also provided herein are spatial array including: a) a plurality ofcapture probes including spatial barcodes and a first plurality ofcapture domains hybridized to a plurality of templated ligationproducts, and b) a plurality of capture probes including spatialbarcodes and a second plurality of capture domains hybridized to aplurality of oligonucleotides from analyte capture agents, where theoligonucleotides include an analyte capture sequence and an analytebinding moiety barcode.

In some embodiments, the capture probes include cleavage domains, uniquemolecular identifiers, one or more functional sequences, or acombination thereof.

In some embodiments, the first plurality of capture domains arehomopolymeric sequences or defined non-homopolymeric sequences. In someembodiments, the second plurality of capture domains are homopolymericsequences or defined non-homopolymeric sequences. In some embodiments,the first plurality of capture domains are poly(T) sequences. In someembodiments, the spatial array includes one or more protein dilutionseries.

Also provided herein are kit including: a) a spatial array including aplurality of capture probes, where the capture probes include spatialbarcodes and where the plurality of capture probes include a firstplurality of first capture domains and a second plurality of secondcapture domains, b) one or more analyte capture agents, c) one or morenucleic acid templated ligation probe pairs, and d) one or more enzymesand buffers for practicing any of the methods described herein.

Also provided herein are methods for determining the spatial location ofa nucleic acid and a protein in a diseased biological sample including:a) providing a spatial array including a first and a second plurality ofcapture probes where each plurality includes a spatial barcode and acapture domain, b) contacting the diseased biological sample with thespatial array, c) contacting the diseased biological sample with: (i) aplurality of analyte capture agents, where an analyte capture agentincludes an analyte binding moiety and an oligonucleotide including ananalyte binding moiety barcode and an analyte capture sequence, wherethe analyte capture sequence includes a sequence complementary to asecond plurality of capture domains, and (ii) a plurality of templatedligation probes, where one of the templated ligation probes includes asequence complementary a first plurality of capture domains, d) bindingthe analyte binding moiety of the analyte capture agent to a targetprotein, e) hybridizing the templated ligation probes to a target RNAand ligating the probes to produce templated ligation products, f)hybridizing the templated ligation products to the first plurality ofcapture domains and the analyte capture sequences of the bound analytecapture agents to the second plurality of capture domains on the spatialarray, and g) determining the sequence or a portion thereof of acaptured ligation product, or a complement, and the sequence of thespatial barcode of the capture probe, or a complement thereof, that isassociated with the ligation product, and the sequence of the analytebinding moiety barcode, or a complement thereof, of the bound analytecapture agent, thereby determining the spatial location of a nucleicacid and the protein in the diseased biological sample.

In some embodiments, the diseased biological sample is a cancerousbiological sample. In some embodiments, the cancerous biological sampleis an ovarian cancer biological sample, a breast cancer biologicalsample, a lung cancer biological sample, or a melanoma. In someembodiments, the breast cancer sample is triple positive breast canceror a ductal cell invasive carcinoma sample.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, patent application, or item ofinformation was specifically and individually indicated to beincorporated by reference. To the extent publications, patents, patentapplications, and items of information incorporated by referencecontradict the disclosure contained in the specification, thespecification is intended to supersede and/or take precedence over anysuch contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 2 is a schematic diagram of an exemplary analyte capture agent.

FIG. 3 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 324 and an analyte capture agent326.

FIGS. 4A-B shows exemplary clustering data for FFPE human lymph nodesections using an eight-plex antibody-oligonucleotide conjugate testpanel. FIG. 4A (RNA) displays clustering with respect to gene expressionof the test panel and FIG. 4B (protein) shows the clustering withrespect to the protein expression of the test panel.

FIG. 5 shows exemplary clustering data of spatial gene (top middle) andprotein expression (top right) in FFPE tonsil tissue sectionssuperimposed on the H&E image (top left). Bottom figures showvisualization of PD-1, Ki67, and CD8A protein markers labeling thefollicular T cells, individual follicles, and suppressor/cytotoxic Tcells, respectively.

FIG. 6 shows exemplary H&E stained FFPE tonsil tissue section (topleft), spatial gene expression data (top middle), and spatial proteinexpression data (top left). FIG. 6 also shows an expanded view of asection of the H&E stained FFPE tonsil tissue section (bottom left) andclustering of gene expression and protein expression of CD8a (bottommiddle) and CD4 (bottom right) superimposed on the section of the H&EFFPE tonsil tissue section.

FIG. 7 shows exemplary spatial gene and protein expression from a subsetof gene and protein expression data from tonsil tissue. A subset ofeighteen different targeted gene and protein expression heat maps areshown.

FIG. 8 shows expanded views of exemplary heat map data for FFPE humantonsil tissue section using a 12-plex antibody-oligonucleotide conjugatetest panel of several genes shown in FIG. 7 .

FIGS. 9A-C show exemplary spatial gene and protein expression in FFPEtriple positive breast cancer tissue sections. FIG. 9A shows an H&Estained FFPE triple positive breast cancer tissue section. FIG. 9B showsrepresentative gene expression clusters and FIG. 9C shows representativeprotein expression clusters. The insets shown in FIGS. 9B and 9C showrepresentative tSNE plots.

FIG. 10 shows an expanded view of Her2 and Vimentin RNA and proteinexpression in FFPE invasive ductal carcinoma tissue sections. Adjacentsections were immunofluorescently stained for Her2 (top) and Vimentin(bottom) demonstrating strong signal within the dashed box region of thebiopsy sample.

FIGS. 11A-C show exemplary spatial gene and protein expression in FFPEinvasive ductal carcinoma tissue sections. FIG. 11B shows representativegene expression clusters and FIG. 11C shows representative proteinexpression clusters superimposed on the H&E stained (FIG. 11A) invasiveductal carcinoma tissue sections.

FIG. 12 shows exemplary spatial gene and protein expression from asubset of the gene and protein expression data from FIGS. 11B-C. Thebiomarkers were used to identify two regions of interest in the invasiveductal carcinoma tissue sample.

FIG. 13 shows exemplary spatial gene and protein expression in FFPEinvasive ductal carcinoma tissue section superimposed on the H&E imageof the ACTA2 and Epcam gene and their corresponding proteins.

FIG. 14A shows an exemplary image of spatially-resolved information ofgene expression of protein tyrosine phosphatase receptor type C (Ptprc)RNA using ligated probes in FFPE mouse spleen tissue under sandwichconfiguration conditions.

FIG. 14B shows an exemplary image of spatially-resolved information ofgene expression of Ptprc RNA using ligated probes in FFPE spleen tissueunder non-sandwich configuration control conditions

FIG. 14C shows an exemplary image of spatially-resolved information ofprotein expression of CD45R using analyte capture agents in FFPE mousespleen tissue under sandwich configuration conditions.

FIG. 14D shows an exemplary image of spatially-resolved information ofprotein expression of CD45R using analyte capture agents in FFPE mousespleen tissue under non-sandwich configuration control conditions.

FIG. 15A shows an exemplary image of spatially-resolved information ofgene expression of tyrosine hydroxylase (Th) RNA using ligated probes inFFPE mouse brain tissue under sandwich configuration conditions.

FIG. 15B shows an exemplary image of spatially-resolved information ofgene expression of Th RNA using ligated probes in FFPE mouse braintissue under non-sandwich configuration control conditions.

FIG. 15C shows an exemplary image of spatially-resolved information ofprotein expression of tyrosine hydroxylase protein (TH) using analytecapture agents in FFPE mouse brain tissue under sandwich configurationconditions.

FIG. 15D shows an exemplary image of spatially-resolved information ofprotein expression of TH using analyte capture agents in FFPE mousebrain tissue under non-sandwich configuration control conditions.

FIG. 16A shows an exemplary image of spatially-resolved information ofgene expression of Ter119 RNA using ligated probes in FFPE mouse embryotorso tissue under sandwich configuration conditions.

FIG. 16B shows an exemplary image of spatially-resolved information ofgene expression of Ter119 RNA using ligated probes in FFPE mouse embryotorso tissue under non-sandwich configuration conditions.

FIG. 16C shows an exemplary image of spatially-resolved information ofprotein expression of Ter119 using an analyte capture agent in FFPEmouse embryo torso tissue under sandwich configuration conditions.

FIG. 16D shows an exemplary image of spatially-resolved information ofprotein expression of Ter119 using analyte capture agents in FFPE mouseembryo torso tissue under non-sandwich configuration conditions.

FIG. 17A shows an exemplary image of spatially-resolved information ofgene expression of Ter119 RNA using ligated probes in FFPE mouse embryohead and upper torso tissue under sandwich configuration conditions.

FIG. 17B shows an exemplary image of spatially-resolved information ofgene expression of Ter119 RNA using ligated probes in FFPE mouse embryohead and upper torso tissue under non-sandwich configuration conditions.

FIG. 17C shows an exemplary image of spatially-resolved information ofprotein expression of Ter119 using analyte capture agents in FFPE mouseembryo head and upper torso tissue under sandwich configurationconditions.

FIG. 17D shows an exemplary image of spatially-resolved information ofprotein expression of Ter119 using an analyte capture agent in FFPEmouse embryo head and upper torso tissue using non-sandwichconfiguration conditions.

FIGS. 18A, 19A, 20A, 21A, and 22A show exemplary images ofspatially-resolved information of gene expression of Mpped1 (FIG. 18A),Tnnc1 (FIG. 19A), Fgf15 (FIG. 20A), Epyc (FIG. 21A), and Serpinale (FIG.22A) RNA using ligated probes in FFPE mouse embryo head and upper torsotissue under sandwich configuration conditions.

FIGS. 18B, 19B, 20B, 21B, and 22B show exemplary images ofspatially-resolved information of gene expression of Mpped1 (FIG. 18B),Tnnc1 (FIG. 19B), Fgf15 (FIG. 20B), Epyc (FIG. 21B), and Serpinale (FIG.22B) RNA using ligated probes in FFPE mouse embryo torso tissue undersandwich configuration conditions.

FIGS. 18C, 19C, 20C, 21C, and 22C show exemplary images ofspatially-resolved information of gene expression of Mpped1 (FIG. 18C),Tnnc1 (FIG. 19C), Fgf15 (FIG. 20C), Epyc (FIG. 21C), and Serpinale (FIG.22C) RNA using ligated probes in FFPE mouse embryo head and upper torsotissue under non-sandwich configuration conditions.

FIGS. 18D, 19D, 20D, 21D, and 22D show exemplary images ofspatially-resolved information of gene expression of Mpped1 (FIG. 18D),Tnnc1 (FIG. 19D), Fgf15 (FIG. 20D), Epyc (FIG. 21D), and Serpinale (FIG.22D) RNA using ligated probes in FFPE mouse embryo torso tissue undernon-sandwich configuration conditions.

FIGS. 18E, 19E, 20E, 21E, and 22E show exemplary images ofspatially-resolved information of protein expression of Mpped1 (FIG.18E), Tnnc1 (FIG. 19E), Fgf15 (FIG. 20E), Epyc (FIG. 21E), and Serpinale(FIG. 22E) using analyte capture agents in FFPE mouse embryo head andupper torso tissue under sandwich configuration conditions.

FIGS. 18F, 19F, 20F, 21F, and 22F show exemplary images ofspatially-resolved information of protein expression of Mpped1 (FIG.18F), Tnnc1 (FIG. 19F), Fgf15 (FIG. 20F), Epyc (FIG. 21F), and Serpinale(FIG. 22F) using analyte capture agents in FFPE mouse embryo head andupper torso tissue under non-sandwich configuration conditions.

FIGS. 18G, 19G, 20G, 21G, and 22G show exemplary images ofspatially-resolved information of protein expression of Mpped1 (FIG.18G), Tnnc1 (FIG. 19G), Fgf15 (FIG. 20G), Epyc (FIG. 21G), and Serpinale(FIG. 22G) using analyte capture agents in FFPE mouse embryo torsotissue under non-sandwich configuration conditions.

FIGS. 18H, 19H, 20H, 21H, and 22H show exemplary images ofspatially-resolved information of gene expression of Mpped1 (FIG. 18I),Tnnc1 (FIG. 19I), Fgf15 (FIG. 20H), Epyc (FIG. 21I), and Serpinale (FIG.22I) RNA using ligated probes in FFPE mouse embryo head and upper torsotissue under non-sandwich configuration conditions in which only RNA wasdetected.

FIGS. 18I, 19I, 20I, 21I, and 22I show exemplary images ofspatially-resolved information of gene expression of Mpped1 (FIG. 18I),Tnnc1 (FIG. 19I), Fgf15 (FIG. 20I), Epyc (FIG. 21I), and Serpinale (FIG.22I) RNA using ligated probes in FFPE mouse embryo torso tissue undernon-sandwich configuration conditions in which only RNA was detected.

FIGS. 23A-E show spatial immune cell infiltration in a breast cancertissue section correlates with pathologist annotations. FIG. 23A showsan H&E stained human breast cancer FFPE tissue section. FIG. 23B showsthe pathologist's annotations. FIG. 23C shows spatial protein expressionof CD3 (e.g., a marker for T cells); FIG. 23D shows spatial proteinexpression of CD8A (e.g., a marker for cytotoxic T cells); and FIG. 23Eshows spatial protein expression for HLA-DR (e.g., a marker for T cellactivation).

FIGS. 24A-D show spatial immune cell infiltration in an ovarian cancerFFPE tissue section correlates with pathologist annotations. FIG. 24Ashows a pathologist's annotation for invasive carcinoma and immunecells. FIG. 24B shows spatial protein expression of CD20 (e.g., a markerfor B cells); FIG. 24C shows spatial protein expression of CD68 (e.g., amarker for monocytes); and FIG. 24D shows spatial protein expression ofCD8A (e.g., a marker for cytotoxic T cells).

FIGS. 25A-D show differential spatial cytotoxic T cell infiltrationwithin different regions of an ovarian cancer FFPE tissue section. FIG.25A shows spatial protein expression of CD8A (e.g., a marker forcytotoxic T cells). FIG. 25B shows highly infiltrated (“hot”, CD3) andnot filtrated (“cold”, CD8) areas of protein expression in the FFPEtissue section shown in FIG. 25A. FIG. 25C shows spatial proteinexpression of HLA-G and FIG. 25D shows spatial protein expression ofIMPG2.

FIG. 26 shows an exemplary spatial array with multiple protein dilutionseries spotted on the spatial array.

FIGS. 27A-D show exemplary spatial gene (FIG. 27B) and proteinexpression (FIG. 27C) expression in lung cancer FFPE tissue. FIG. 27Ashows an H&E stained lung cancer FFPE tissue and FIG. 27D shows a HLA-DRprotein spatial UMI plot.

FIGS. 28A-D show exemplary spatial gene (FIG. 28B) and proteinexpression (FIG. 28C) expression in melanoma FFPE tissue. FIG. 28A showsan H&E stained melanoma cancer tissue and FIG. 28D shows a HLA-DRprotein spatial UMI plot.

FIGS. 29A-C show exemplary Vimentin antibody immunofluorescence stainingand DAPI staining (FIG. 29A), exemplary spatial protein expression (FIG.29B), and exemplary Vimentin spatial protein expression (FIG. 29C) in agrade II invasive ductal carcinoma FFPE breast cancer tissue section.

DETAILED DESCRIPTION

The present disclosure features methods, compositions, and kits forspatial analysis of biological samples. More specifically, the presentdisclosure features methods, compositions, and kits for both spatialgene expression and spatial protein expression in a biological sample.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10x Genomics Support Documentationwebsite, and can be used herein in any combination, and each of which isincorporated herein by reference in their entireties. Furthernon-limiting aspects of spatial analysis methodologies and compositionsare described herein. Some general terminology that may be used in thisdisclosure can be found in Section (I)(b) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. Typically, a “barcode”is a label, or identifier, that conveys or is capable of conveyinginformation (e.g., information about an analyte in a sample, a bead,and/or a capture probe). A barcode can be part of an analyte, orindependent of an analyte. A barcode can be attached to an analyte. Aparticular barcode can be unique relative to other barcodes. For thepurpose of this disclosure, an “analyte” can include any biologicalsubstance, structure, moiety, or component to be analyzed. The term“target” can similarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a ligation product or an analyte capture agent (e.g., anoligonucleotide-conjugated antibody), such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Generation of capture probes can be achieved by any appropriate method,including those described in Section (II)(d)(ii) of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

FIG. 1 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 102 is optionally coupledto a feature 101 by a cleavage domain 103, such as a disulfide linker.The capture probe can include a functional sequence 104 that are usefulfor subsequent processing. The functional sequence 104 can include allor a part of sequencer specific flow cell attachment sequence (e.g., aP5 or P7 sequence), all or a part of a sequencing primer sequence,(e.g., a R1 primer binding site, a R2 primer binding site), orcombinations thereof. The capture probe can also include a spatialbarcode 105. The capture probe can also include a unique molecularidentifier (UMI) sequence 106. While FIG. 1 shows the spatial barcode105 as being located upstream (5′) of UMI sequence 106, it is to beunderstood that capture probes wherein UMI sequence 106 is locatedupstream (5′) of the spatial barcode 105 is also suitable for use in anyof the methods described herein. The capture probe can also include acapture domain 107 to facilitate capture of a target analyte. In someembodiments, the capture probe comprises one or more additionalfunctional sequences that can be located, for example between thespatial barcode 105 and the UMI sequence 106, between the UMI sequence106 and the capture domain 107, or following the capture domain 107. Thecapture domain can have a sequence complementary to a sequence of anucleic acid analyte. The capture domain can have a sequencecomplementary to a connected probe described herein. The capture domaincan have a sequence complementary to a capture handle sequence presentin an analyte capture agent. The capture domain can have a sequencecomplementary to a splint oligonucleotide. Such splint oligonucleotide,in addition to having a sequence complementary to a capture domain of acapture probe, can have a sequence of a nucleic acid analyte, a sequencecomplementary to a portion of a connected probe described herein, and/ora capture handle sequence described herein.

The functional sequences can generally be selected for compatibilitywith any of a variety of different sequencing systems, e.g., Ion TorrentProton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore,etc., and the requirements thereof. In some embodiments, functionalsequences can be selected for compatibility with non-commercializedsequencing systems. Examples of such sequencing systems and techniques,for which suitable functional sequences can be used, include (but arenot limited to) Ion Torrent Proton or PGM sequencing, Illuminasequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 105 and functional sequences104 is common to all of the probes attached to a given feature. In someembodiments, the UMI sequence 106 of a capture probe attached to a givenfeature is different from the UMI sequence of a different capture probeattached to the given feature.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence. In some embodiments, the analyte capture agentincludes a capture agent barcode domain that is conjugated or otherwiseattached to the analyte binding moiety. In some embodiments, the captureagent barcode domain is covalently-linked to the analyte binding moiety.In some embodiments, a capture agent barcode domain is a nucleic acidsequence. In some embodiments, a capture agent barcode domain includesan analyte binding moiety barcode and an analyte capture sequence.

In some embodiments, analyte capture agents are capable of binding toanalytes present inside a cell. In some embodiments, analyte captureagents are capable of binding to cell surface analytes that can include,without limitation, a receptor, an antigen, a surface protein, atransmembrane protein, a cluster of differentiation protein, a proteinchannel, a protein pump, a carrier protein, a phospholipid, aglycoprotein, a glycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, an extracellular matrix protein, aposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation)state of a cell surface protein, a gap junction, and an adherensjunction. In some embodiments, the analyte capture agents are capable ofbinding to cell surface analytes that are post-translationally modified.In such embodiments, analyte capture agents can be specific for cellsurface analytes based on a given state of posttranslationalmodification (e.g., phosphorylation, glycosylation, ubiquitination,nitrosylation, methylation, acetylation or lipidation), such that a cellsurface analyte profile can include posttranslational modificationinformation of one or more analytes.

As used herein, the term “analyte binding moiety” refers to a moleculeor moiety capable of binding to a macromolecular constituent (e.g., ananalyte, e.g., a biological analyte). In some embodiments of any of thespatial profiling methods described herein, the analyte binding moietyof the analyte capture agent that binds to a biological analyte caninclude, but is not limited to, an antibody, or an epitope bindingfragment thereof, a cell surface receptor binding molecule, a receptorligand, a small molecule, a bi-specific antibody, a bi-specific T-cellengager, a T-cell receptor engager, a B-cell receptor engager, apro-body, an aptamer, a monobody, an affimer, a darpin, and a proteinscaffold, or any combination thereof. The analyte binding moiety canbind to the macromolecular constituent (e.g., analyte) with highaffinity and/or with high specificity. The analyte binding moiety caninclude a nucleotide sequence (e.g., an oligonucleotide), which cancorrespond to at least a portion or an entirety of the analyte bindingmoiety. The analyte binding moiety can include a polypeptide and/or anaptamer (e.g., a polypeptide and/or an aptamer that binds to a specifictarget molecule, e.g., an analyte). The analyte binding moiety caninclude an antibody or antibody fragment (e.g., an antigen-bindingfragment) that binds to a specific analyte (e.g., a polypeptide).

In some embodiments, an analyte binding moiety of an analyte captureagent includes one or more antibodies or antigen binding fragmentsthereof. The antibodies or antigen binding fragments including theanalyte binding moiety can specifically bind to a target analyte. Insome embodiments, the analyte is a protein (e.g., a protein on a surfaceof the biological sample (e.g., a cell) or an intracellular protein). Insome embodiments, a plurality of analyte capture agents comprising aplurality of analyte binding moieties bind a plurality of analytespresent in a biological sample. In some embodiments, the plurality ofanalytes includes a single species of analyte (e.g., a single species ofpolypeptide). In some embodiments in which the plurality of analytesincludes a single species of analyte, the analyte binding moieties ofthe plurality of analyte capture agents are the same. In someembodiments in which the plurality of analytes includes a single speciesof analyte, the analyte binding moieties of the plurality of analytecapture agents are the different (e.g., members of the plurality ofanalyte capture agents can have two or more species of analyte bindingmoieties, wherein each of the two or more species of analyte bindingmoieties binds a single species of analyte, e.g., at different bindingsites). In some embodiments, the plurality of analytes includes multipledifferent species of analyte (e.g., multiple different species ofpolypeptides).

As used herein, the term “analyte binding moiety barcode” refers to abarcode that is associated with or otherwise identifies the analytebinding moiety. In some cases, an analyte binding moiety barcode (orportion thereof) may be able to be removed (e.g., cleaved) from theanalyte capture agent. In some embodiments, by identifying an analytebinding moiety and its associated analyte binding moiety barcode, theanalyte to which the analyte binding moiety binds can also beidentified. An analyte binding moiety barcode can be a nucleic acidsequence of a given length and/or sequence that is associated with theanalyte binding moiety. An analyte binding moiety barcode can generallyinclude any of the variety of aspects of barcodes described herein. Forexample, an analyte capture agent that is specific to one type ofanalyte can have coupled thereto a first capture agent barcode domain(e.g., that includes a first analyte binding moiety barcode), while ananalyte capture agent that is specific to a different analyte can have adifferent capture agent barcode domain (e.g., that includes a secondbarcode analyte binding moiety barcode) coupled thereto. In someaspects, such a capture agent barcode domain can include an analytebinding moiety barcode that permits identification of the analytebinding moiety to which the capture agent barcode domain is coupled. Theselection of the capture agent barcode domain can allow significantdiversity in terms of sequence, while also being readily attachable tomost analyte binding moieties (e.g., antibodies or aptamers) as well asbeing readily detected, (e.g., using sequencing or array technologies).Additional description of analyte capture agents can be found in Section(II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. Patent

Application Publication No. 2020/0277663.

In some embodiments, the capture agent barcode domain of an analytecapture agent includes an analyte capture sequence. As used herein, theterm “analyte capture sequence” refers to a region or moiety configuredto hybridize to, bind to, couple to, or otherwise interact with acapture domain of a capture probe. In some embodiments, an analytecapture sequence includes a nucleic acid sequence that is complementaryto or substantially complementary to the capture domain of a captureprobe such that the analyte capture sequence hybridizes to the capturedomain of the capture probe. In some embodiments, an analyte capturesequence comprises a poly(A) nucleic acid sequence that hybridizes to acapture domain that comprises a poly(T) nucleic acid sequence. In someembodiments, an analyte capture sequence comprises a poly(T) nucleicacid sequence that hybridizes to a capture domain that comprises apoly(A) nucleic acid sequence. In some embodiments, an analyte capturesequence comprises a non-homopolymeric nucleic acid sequence thathybridizes to a capture domain that comprises a non-homopolymericnucleic acid sequence that is complementary (or substantiallycomplementary) to the non-homopolymeric nucleic acid sequence of theanalyte capture region.

FIG. 2 is a schematic diagram of an exemplary analyte capture agent 202comprised of an analyte binding moiety 204 and a capture agent barcodedomain 208. An analyte binding moiety 204 is a molecule capable ofbinding to an analyte 206 and interacting with a spatially-barcodedcapture probe. The analyte binding moiety can bind to the analyte 206with high affinity and/or with high specificity. The analyte captureagent can include a capture agent barcode domain 208, a nucleotidesequence (e.g., an oligonucleotide), which can hybridize to at least aportion or an entirety of a capture domain of a capture probe. Theanalyte binding moiety 204 can include a polypeptide and/or an aptamer(e.g., an oligonucleotide or peptide molecule that binds to a specifictarget analyte). The analyte binding moiety 204 can include an antibodyor antibody fragment (e.g., an antigen-binding fragment).

FIG. 3 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 324 and an analyte capture agent326. The feature-immobilized capture probe 324 can include a spatialbarcode 308 as well as one or more functional sequences 306 and 310, asdescribed elsewhere herein. The capture probe can also include a capturedomain 312 that is capable of binding to an analyte capture agent 326.The analyte capture agent 326 can include a functional sequence 318,capture agent barcode domain 316, and an analyte capture sequence 314that is capable of binding to the capture domain 312 of the captureprobe 324. The analyte capture agent can also include a linker 320 thatallows the capture agent barcode domain 316 to couple to the analytebinding moiety 322.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a ligation product or an analyte captureagent), or a portion thereof), or derivatives thereof (see, e.g.,Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663 regarding extended capture probes). In somecases, capture probes may be configured to form ligation products with atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent, or portion thereof), thereby creating ligationsproducts that serve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Exemplary methods for identifying spatial information of biologicaland/or medical importance can be found in U.S. Patent ApplicationPublication No. 2021/0140982A1, U.S. Patent Application No.2021/0198741A1, and/or U.S. Patent Application No. 2021/0199660.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a ligation product. In some instances, the two oligonucleotideshybridize to sequences that are not adjacent to one another. Forexample, hybridization of the two oligonucleotides creates a gap betweenthe hybridized oligonucleotides. In some instances, a polymerase (e.g.,a DNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). The released ligation product can then becaptured by capture probes (e.g., instead of direct capture of ananalyte) on an array, optionally amplified, and sequenced, thusdetermining the location and optionally the abundance of the analyte inthe biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in WO 2021/102003and/or U.S. patent application Ser. No. 16/951,854, each of which isincorporated herein by reference in their entireties.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2021/102039and/or U.S. patent application Ser. No. 16/951,864, each of which isincorporated herein by reference in their entireties.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, WO2021/102005, and/or U.S. patent application Ser. No. 16/951,843, each ofwhich is incorporated herein by reference in their entireties. Fiducialmarkers can be used as a point of reference or measurement scale foralignment (e.g., to align a sample and an array, to align twosubstrates, to determine a location of a sample or array on a substraterelative to a fiducial marker) and/or for quantitative measurements ofsizes and/or distances.

Multiplex Gene Expression and Protein Analysis

Understanding both gene and protein expression in biological systems canbe helpful for gaining insights into normal, developing, and diseasedtissues. While single cell RNA-seq (scRNA-seq) makes it possible toobtain high-resolution gene expression measurements, the techniquerequires cells to be dissociated, thereby losing anatomical andorganizational information. Similarly, numerous protein detectiontechniques are known and can provide spatial information of proteins ina biological sample, however, methods of simultaneously detecting levelsof gene expression (e.g., mRNA) of the protein, or even the entiretranscriptome are still needed.

Thus, disclosed herein are “multi-omics” approaches that can provide apowerful complement to traditional methodologies, enabling a greaterunderstanding of cellular heterogeneity and organization withinbiological samples. The combination of protein detection using analytecapture agents on a spatial array allows for the simultaneousexamination of protein and gene expression from the same biologicalsample (e.g., tissue section). For example, an array comprising captureprobes (e.g., any of the capture probes described herein) can becontacted with a biological sample, a plurality of templated ligationprobes, and a plurality of analyte capture agents that result insimultaneous gene and protein expression analysis.

In some embodiments, the plurality of templated ligation probes includea pair of probes for a target nucleic acid (e.g., DNA, RNA). The probesare complementary to portions of the target nucleic acid, however, whenboth probes hybridize to the target nucleic acid a gap is presentbetween the two probes. In some embodiments, the gap is ligated, therebygenerating a templated ligation product (e.g., DNA or RNA templatedligation product). In some embodiments, one of the pair of probesincludes a flanking sequence complementary to a capture domain of thearray. In some embodiments, the sequence complementary to the capturedomain of the templated ligation product hybridizes to the capturedomain of the capture probe.

In some embodiments, analyte capture agents, as described herein, canalso be contacted with the biological sample. In some embodiments, theanalyte capture agents are contacted with the biological sample beforethe biological sample is contacted with an array. In some embodiments,the analyte capture agents are contacted with the biological sampleafter the biological sample is contacted with the array. In someembodiments, the analyte binding moiety of the analyte capture agentinteracts (e.g., binds) to an analyte (e.g., protein) in a biologicalsample. In some embodiments, the analyte binding moiety is an antibodyor antigen-binding fragment.

Analyte capture agents can also include a conjugated oligonucleotidethat can comprise one or more domains. For example, the conjugatedoligonucleotide can include an analyte binding moiety barcode and ananalyte capture sequence. In some embodiments, the analyte bindingmoiety barcode, or a complement thereof, refers to (e.g., identifies) abarcode that is associated with or otherwise identifies the analytebinding moiety. In some embodiments, the conjugated oligonucleotide caninclude an analyte capture sequence. In some embodiments, the analytecapture sequence is capable of interacting with (e.g., hybridizing) to acapture domain of a capture probe on a substrate.

In some embodiments, the templated ligation probes are allowed to bindthe target nucleic acid before the analyte capture agents are deliveredto the biological sample. In some embodiments, the templated ligationprobes can be ligated together before, concurrently, or after theanalyte capture agents are delivered to the biological sample. In someembodiments, the analyte capture agents are delivered to the biologicalsample and the analyte binding moiety is allowed to bind the targetanalyte (e.g., protein) before the templated ligation probes aredelivered. In some embodiments, the analyte capture agents are deliveredto the biological sample and the analyte capture sequence is blocked(e.g., blocked by any of the methods described herein). In someembodiments, the analyte capture sequence of the analyte capture agentsis unblocked (e.g., unblocked by any of the methods described herein)before, concurrently, or after the templated ligation probes (e.g., RNAtemplated ligation probes) are delivered and/or before, concurrently, orafter the templated ligation probes are ligated together.

Thus, provided herein are methods for determining the spatial locationof a nucleic acid and a protein from a biological sample including: a)providing a spatial array including a first and second plurality ofcapture probes where each plurality includes a spatial barcode and acapture domain, b) contacting the spatial array with a biologicalsample, c) contacting the biological sample with (i) a plurality ofanalyte capture agents, where an analyte capture agent includes ananalyte binding moiety and an oligonucleotide including an analytebinding moiety barcode and an analyte capture sequence, where theanalyte capture sequence includes a sequence complementary to a secondplurality of capture domains, and (ii) a plurality of templated ligationprobes, where one of the templated ligation probes includes a sequencecomplementary a first plurality of capture domains, d) binding theanalyte binding moiety of the analyte capture agent to a target protein,e) hybridizing the templated ligation probes to a target nucleic acidand ligating the probes to produce ligation products, f) hybridizing theligation products to the first plurality of capture domains and theanalyte capture sequences of the bound analyte capture agents to thesecond plurality of capture domains on the spatial array, and g)determining the sequence or a portion thereof of a captured ligationproduct, or a complement thereof, and the sequence of the spatialbarcode of the capture probe, or a complement thereof, that isassociated with the ligation product, and the sequence of the analytebinding moiety barcode, or a complement thereof, of the bound analytecapture agent, thereby determining the spatial location of a nucleicacid and the protein from the biological sample.

Also provided herein are methods for determining the spatial location ofa nucleic acid and a protein in a diseased biological sample including:a) providing a spatial array including a first and a second plurality ofcapture probes where each plurality includes a spatial barcode and acapture domain, b) contacting the diseased biological sample with thespatial array, c) contacting the diseased biological sample with: (i) aplurality of analyte capture agents, where an analyte capture agentincludes an analyte binding moiety and an oligonucleotide including ananalyte binding moiety barcode and an analyte capture sequence, wherethe analyte capture sequence includes a sequence complementary to asecond plurality of capture domains, and (ii) a plurality of templatedligation probes, where one of the templated ligation probes includes asequence complementary a first plurality of capture domains, d) bindingthe analyte binding moiety of the analyte capture agent to a targetprotein, e) hybridizing the templated ligation probes to a target RNAand ligating the probes to produce templated ligation products, f)hybridizing the templated ligation products to the first plurality ofcapture domains and the analyte capture sequences of the bound analytecapture agents to the second plurality of capture domains on the spatialarray, and g) determining the sequence or a portion thereof of acaptured ligation product, or a complement, and the sequence of thespatial barcode of the capture probe, or a complement thereof, that isassociated with the ligation product, and the sequence of the analytebinding moiety barcode, or a complement thereof, of the bound analytecapture agent, thereby determining the spatial location of a nucleicacid and the protein in the diseased biological sample.

In some embodiments, the nucleic acid is RNA. In some embodiments, theRNA is mRNA. In some embodiments, the nucleic acid is DNA.

In some embodiments, the diseased biological sample is a cancerousbiological sample. In some embodiments, the cancerous biological sampleis an ovarian cancer biological sample or a breast cancer biologicalsample. In some embodiments, the breast cancer sample is triple positivebreast cancer. In some embodiments, the breast cancer is invasive ductalcell carcinoma breast cancer. In some embodiments, the invasive ductalcell carcinoma is grade II invasive ductal carcinoma. In someembodiments, the invasive ductal cell carcinoma is grade III invasiveductal carcinoma. In some embodiments, the breast cancer is invasivelobular carcinoma. In some embodiments, the cancerous biological sampleis lung cancer. In some embodiments, the cancerous biological sample ismelanoma. In some embodiments, the cancerous biological sample is coloncancer. In some embodiments, the cancerous biological sample isglioblastoma. In some embodiments, the cancerous biological sample isprostate cancer.

In some embodiments, the capture probes include unique molecularidentifiers, functional sequences, or combinations thereof.

In some embodiments, the first plurality of capture domains arehomopolymeric sequences. In some embodiments, the first plurality ofcapture domains comprise poly(T) sequences. In some embodiments, thefirst plurality of capture domains are defined non-homopolymericsequences. In some embodiments, the first plurality of capture domainsincludes a degenerate sequence. In some embodiments, the first pluralityof capture domains includes a fixed sequence. For example, the firstplurality of capture domains can comprises one of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. Insome embodiments, the second plurality of capture domains arehomopolymeric sequences. In some embodiments, the second plurality ofcapture domains are defined non-homopolymeric sequences. In someembodiments, the second plurality of capture domains comprise poly(T)sequences. In some embodiments, the second plurality of capture domainsincludes a degenerate sequence. In some embodiments, the secondplurality of capture domains includes a fixed sequence. For example, thesecond plurality of capture domains can comprise one of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

In some embodiments, the first plurality of capture domains and thesecond plurality of capture domains include the same sequence. In someembodiments, the first plurality of capture domains and the secondplurality of capture domains include different sequences.

Generally, the methods of the present disclosure can be used with anybiological sample (e.g., any biological sample described herein). Insome embodiments, the biological sample is a tissue section. In someembodiments, the biological sample is a tissue sample. In someembodiments, the biological sample is a fresh-frozen biological sample.In some embodiments, the biological sample is a fixed biological sample(e.g., formalin-fixed paraffin embedded (FFPE), paraformaldehyde,acetone, or methanol). In some embodiments, the biological sample is anFFPE sample. In some embodiments, the biological sample is an FFPEtissue section. In some embodiments, the tissue sample is a tumorsample. In some embodiment, the tissue section is a tumor tissuesection. In some embodiments, the tumor tissue section is a fixed tumortissue section (e.g., a formal-fixed paraffin-embedded tumor tissuesection). In some embodiments, the tumor sample comprises one or morecancer tumors. Numerous types of cancer are known in the art. In someembodiments, the tissue sample is derived from a biopsy sample. In someembodiments, the tissue sample is derived from a whole rodent embryo. Insome embodiments, the tissue is selected from, but not limited to, braintissue, breast tissue, colon tissue, heart tissue, lung tissue, spleentissue, testes tissue, inflamed tonsil tissue, cervix tissue, and lymphnode tissue.

In some embodiments, an FFPE sample is deparaffinized and decrosslinkedprior to delivering a plurality of templated ligation probes (e.g., RNAtemplated ligation probes) and analyte capture agents. In someembodiments, an FFPE biological sample is deparaffinized anddecrosslinked before step (b). For example, the paraffin-embeddingmaterial can be removed (e.g., deparaffinization) from the biologicalsample (e.g., tissue section) by incubating the biological sample in anappropriate solvent (e.g., xylene), followed by a series of rinses(e.g., ethanol of varying concentrations), and rehydration in water. Insome embodiments, the biological sample can be dried followingdeparaffinization. In some embodiments, after the step of drying thebiological sample, the biological sample can be stained (e.g., H&Estain, any of the variety of stains described herein).

In some embodiments, the method includes staining the biological sample.In some embodiments, the staining includes the use of hematoxylin andeosin. In some embodiments, a biological sample can be stained using anynumber of biological stains, including but not limited to, acridineorange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI,eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains,iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red,osmium tetroxide, propidium iodide, rhodamine, or safranin.

The biological sample can be stained using known staining techniques,including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's,Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan,Wright's, and/or Periodic Acid Schiff (PAS) staining techniques. PASstaining is typically performed after formalin or acetone fixation.

In some embodiments, the staining includes the use of a detectable labelselected from the group consisting of a radioisotope, a fluorophore, achemiluminescent compound, a bioluminescent compound, or a combinationthereof.

In some embodiments, the biological sample is imaged after staining thebiological sample. In some embodiments, the biological sample is imagedprior to staining the biological sample. In some embodiments, thebiological sample is visualized or imaged using bright field microscopy.In some embodiments, the biological sample is visualized or imaged usingfluorescence microscopy. Additional methods of visualization and imagingare known in the art. Non-limiting examples of visualization and imaginginclude expansion microscopy, bright field microscopy, dark fieldmicroscopy, phase contrast microscopy, electron microscopy, fluorescencemicroscopy, reflection microscopy, interference microscopy and confocalmicroscopy. In some embodiments, the sample is stained and imaged priorto adding the first and/or second primer to the biological sample on thearray.

After a fixed (e.g., FFPE, PFA, acetone, methanol) biological sample hasundergone deparaffinization, the fixed (e.g., FFPE, PFA) biologicalsample can be further processed.

For example, fixed (e.g., FFPE, PFA) biological samples can be treatedto remove crosslinks (e.g., formaldehyde-induced crosslinks (e.g.,decrosslinking)). In some embodiments, decrosslinking the crosslinks(e.g., formaldehyde-induced crosslinks) in the fixed (e.g., FFPE, PFA)biological sample can include treating the sample with heat. In someembodiments, decrosslinking the formaldehyde-induced crosslinks caninclude performing a chemical reaction. In some embodiments, decrosslinking the formaldehyde-induced crosslinks, can include treating thesample with a permeabilization reagent. In some embodiments,decrosslinking the formaldehyde-induced crosslinks can include heat, achemical reaction, and/or permeabilization reagents. In someembodiments, decrosslinking crosslinks (e.g., formaldehyde-inducedcrosslinks) can be performed in the presence of a buffer. In someembodiments, the buffer is Tris-EDTA (TE) buffer (e.g., TE buffer forFFPE biological samples). In some embodiments, the buffer is citratebuffer (e.g., citrate buffer for FFPE biological samples). In someembodiments, the buffer is Tris-HCl buffer (e.g., Tris-HCl buffer forPFA fixed biological samples). In some embodiments, the buffer (e.g., TEbuffer, Tris-HCl buffer) has a pH of about 5.0 to about 10.0 and atemperature between about 60° C. to about 100° C.

In some embodiments, the biological sample is permeabilized (e.g.,permeabilized by any of the methods described herein). In someembodiments, the permeabilization is an enzymatic permeabilization. Insome embodiments, the permeabilization is a chemical permeabilization.In some embodiments, the biological sample is permeabilized beforedelivering the RNA templated ligation probes and analyte capture agentsto the biological sample. In some embodiments, the biological sample ispermeabilized at the same time as the RNA templated ligation probes andanalyte capture agents are delivered to the biological sample. In someembodiments, the biological sample is permeabilized after the RNAtemplated ligation probes and analyte capture agents are delivered tothe biological sample. In some embodiments, hybridizing the RNAtemplated ligation products to the second capture domains and theanalyte capture sequences of the bound analyte capture agents to thefirst capture domains further comprises permeabilizing the biologicalsample.

In some embodiments, the biological sample is permeabilized from about30 to about 120 minutes, from about 40 to about 110 minutes, from about50 to about 100 minutes, from about 60 to about 90 minutes, or fromabout 70 to 80 minutes. In some embodiments, the biological samples ispermeabilized about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 90, about95, about 100, about 105, about 110, about 115, or about 130 minutes.

In some embodiments, the permeabilization buffer comprises urea. In someembodiments, the urea is at a concentration of about 0.5M to 3.0M. Insome embodiments, the concentration of the urea is about 0.5, 1.0, 1.5,2.0, 2.5, or about 3.0M. In some embodiments, the permeabilizationbuffer includes a detergent. In some embodiments, the detergent issarkosyl. In some embodiments, the sarkosyl is present at about 2% toabout 10% (v/v). In some embodiments, the sarkosyl is present at about3%, 4%, 5%, 6%, 7%, 8%, or 9% (v/v). In some embodiments, thepermeabilization buffer comprises polyethylene glycol (PEG). In someembodiments, the PEG is from about PEG 2K to about PEG 16K. In someembodiments, the PEG is PEG 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, 11K,12K, 13K, 14K, 15K, or 16K. In some embodiments, the PEG is present at aconcentration from about 2% to 25%, from about 4% to about 23%, fromabout 6% to about 21%, or from about 8% to about 20% (v/v).

In some embodiments, the method includes a step of permeabilizing thebiological sample (e.g., a tissue section). For example, the biologicalsample can be permeabilized to facilitate transfer of the extendedproducts to the capture probes on the array. In some embodiments, thepermeabilizing includes the use of an organic solvent (e.g., acetone,ethanol, and methanol), a detergent (e.g., saponin, Triton X100™,Tween-20™, or sodium dodecyl sulfate (SDS)), and an enzyme (anendopeptidase, an exopeptidase, a protease), or combinations thereof. Insome embodiments, the permeabilizing includes the use of anendopeptidase, a protease, SDS, polyethylene glycol tert-octylphenylether, polysorbate 80, and polysorbate 20, N-lauroylsarcosine sodiumsalt solution, saponin, Triton X100™, Tween-20™, or combinationsthereof. In some embodiments, the endopeptidase is pepsin. In someembodiments, the endopeptidase is Proteinase K. Additional methods forsample permeabilization are described, for example, in Jamur et al.,Method Mol. Biol. 588:63-66, 2010, the entire contents of which areincorporated herein by reference.

The methods provided herein can also include antibody staining. In someembodiment, antibody staining includes the use of an antibody stainingbuffer. In some embodiments, the antibody staining buffer (e.g., aPBS-based buffer) includes a detergent (e.g., Tween-20, SDS, sarkosyl).In some embodiments, the antibody staining buffer includes a serum, suchas for example, a goat serum. In some embodiments, the goat serum isfrom about 1% to about 10% (v/v), from about 2% to about 9% (v/v), fromabout 3% to about 8% (v/v), or about 4% to about 7% (v/v). In someembodiments, the antibody staining buffer includes dextran sulfate. Insome embodiments, the dextran sulfate is at a concentration of about 1mg/ml to about 20 mg/ml, from about 5 mg/ml to about 15 mg/ml, or fromabout 8 mg/ml to about 12 mg/ml.

The methods provided herein can also utilize blocking probes to blockthe non-specific binding (e.g., hybridization) of the analyte capturesequence and the capture domain of a capture probe on an array. In someembodiments, following contact between the biological sample and thearray, the biological sample is contacted with a plurality of analytecapture agents, where an analyte capture agent includes an analytecapture sequence that is reversibly blocked with a blocking probe. Insome embodiments, the analyte capture sequence is reversibly blockedwith more than one blocking probe (e.g., 2, 3, 4, or more blockingprobes). In some embodiments, the analyte capture agent is blocked priorto binding the target analyte (e.g., a target protein).

In some embodiments, the oligonucleotide of the analyte capture agent(e.g., analyte capture sequence) is blocked by a blocking probe. In someembodiments, blocking probes are hybridized to the analyte capturesequence of the analyte capture agents before introducing the analytecapture agents to a biological sample. In some embodiments, blockingprobes are hybridized to the analyte capture sequence of the analytecapture agents after introducing the analyte capture agents to thebiological sample. In such embodiments, the capture domain can also beblocked to prevent non-specific binding, and/or to control the time ofbinding, between the analyte capture sequence and the capture domain. Insome embodiments, the blocking probes can be alternatively oradditionally introduced during staining of the biological sample. Insome embodiments, the analyte capture sequence is blocked prior tobinding to the capture domain, where the blocking probe includes asequence complementary or substantially complementary to the analytecapture sequence.

In some embodiments, the analyte capture sequence is blocked with oneblocking probe. In some embodiments, the analyte capture sequence isblocked with two blocking probes. In some embodiments, the analytecapture sequence is blocked with more than two blocking probes (e.g., 3,4, 5, or more blocking probes). In some embodiments, a blocking probe isused to block the free 3′ end of the analyte capture sequence. In someembodiments, a blocking probe is used to block the 5′ end of the analytecapture sequence. In some embodiments, two blocking probes are used toblock both 5′ and 3′ ends of the analyte capture sequence. In someembodiments, both the analyte capture sequence and the capture probedomain are blocked.

In some embodiments, the blocking probes can differ in length and/orcomplexity. In some embodiments, the blocking probe can include anucleotide sequence of about 8 to about 24 nucleotides in length (e.g.,about 8 to about 22, about 8 to about 20, about 8 to about 18, about 8to about 16, about 8 to about 14, about 8 to about 12, about 8 to about10, about 10 to about 24, about 10 to about 22, about 10 to about 20,about 10 to about 18, about 10 to about 16, about 10 to about 14, about10 to about 12, about 12 to about 24, about 12 to about 22, about 12 toabout 20, about 12 to about 18, about 12 to about 16, about 12 to about14, about 14 to about 24, about 14 to about 22, about 14 to about 20,about 14 to about 18, about 14 to about 16, about 16 to about 24, about16 to about 22, about 16 to about 20, about 16 to about 18, about 18 toabout 24, about 18 to about 22, about 18 to about 20, about 20 to about24, about 20 to about 22, or about 22 to about 24 nucleotides inlength).

In some embodiments, the blocking probe is removed prior to hybridizingthe analyte capture sequence of the oligonucleotide of the analytecapture sequence to the first capture domain. For example, once theblocking probe is released from the analyte capture sequence, theanalyte capture sequence can bind to the first capture domain on thearray. In some embodiments, blocking the analyte capture sequencereduces non-specific background staining. In some embodiments, blockingthe analyte capture sequence allows for control over when to allow thebinding of the analyte capture sequence to the capture domain of acapture probe during a spatial workflow, thereby controlling the time ofcapture of the analyte capture sequence on the array. In someembodiments, the blocking probes are reversibly bound, such that theblocking probes can be removed from the analyte capture sequence duringor after the time that analyte capture agents are in contact with thebiological sample. In some embodiments, the blocking probe can beremoved with RNAse treatment (e.g., RNAse H treatment). In someembodiments, the blocking probes are removed by increasing thetemperature (e.g., heating) the biological sample. In some embodiments,the blocking probes are removed enzymatically (e.g., cleaved). In someembodiments, the blocking probes are removed by a USER enzyme. In someembodiments, the blocking probes are removed by an endonuclease. In someembodiments, the endonuclease is endonuclease IV. In some embodiments,the endonuclease is endonuclease V.

In some embodiments, the determining in step (g) includes a) extendingthe captured ligation products and the captured oligonucleotides of theanalyte capture agents, wherein the extension products comprise thespatial barcode or a complement thereof, b) releasing the extensionproducts, or complements thereof, from the spatial array, c) producing alibrary from the released extension products or complements thereof, andd) sequencing the library. In some embodiments, extension (e.g.,extension of captured nucleic acid ligation products and the capturedoligonucleotides of the analyte capture agents and/or extension of theplurality of captures probes) is performed with a polymerase (e.g., anysuitable polymerase, e.g., T4 polymerase).

In some embodiments, the released extension products can be prepared fordownstream applications, such as generation of a sequencing library andnext-generation sequencing. Producing sequencing libraries are known inthe art. For example, the released extension products can be purifiedand collected for downstream amplification steps. The released extensionproducts can be amplified using PCR, where primer binding sites flankthe spatial barcode and ligation product or analyte binding moietybarcode, or complements thereof, generating a library associated with aparticular spatial barcode. In some embodiments, the library preparationcan be quantitated and/or quality controlled to verify the success ofthe library preparation steps. The library amplicons are sequenced andanalyzed to decode spatial information and the ligation product oranalyte binding moiety barcode, or complements thereof.

Alternatively or additionally, the amplicons can then be enzymaticallyfragmented and/or size-selected in order to provide for desired ampliconsize. In some embodiments, when utilizing an Illumina® librarypreparation methodology, for example, P5 and P7, sequences can be addedto the amplicons thereby allowing for capture of the library preparationon a sequencing flowcell (e.g., on Illumina sequencing instruments).Additionally, i7 and i5 can index sequences be added as sample indexesif multiple libraries are to be pooled and sequenced together. Further,Read 1 and Read 2 sequences can be added to the library preparation forsequencing purposes. The aforementioned sequences can be added to alibrary preparation sample, for example, via End Repair, A-tailing,Adaptor Ligation, and/or PCR. The cDNA fragments can then be sequencedusing, for example, paired-end sequencing using TruSeq Read 1 and TruSeqRead 2 as sequencing primer sites, although other methods are known inthe art.

In some embodiments, the determining in step (g) can include apre-amplification step. For example, a complementary strand to theextended RNA ligation products and/or the extension product of thecaptured oligonucleotides of the analyte capture agents the step can begenerated and further include a pre-amplification step of the extensionproducts or complements thereof (e.g., extended products) prior tolibrary production (e.g., RTL library production; capturedoligonucleotide of the analyte capture agent production).

Compositions

Also provided herein are spatial arrays, including spatial arraysdescribed in the methods herein, that include a dilution series ofprotein standards directly on the array. In general, proteinquantification with antibodies can be difficult due to the varyingaffinity antibodies have for their protein targets. In order toaccurately quantify protein abundance with antibodies, standard curveswith the protein of interest (e.g., similar to an ELISA assay) can beapplied to a spatial array in parallel with spatial proteomic analysis.In some embodiments, a protein standard is spotted on the array (e.g.,on top of the features of the array). In some embodiments, more than oneprotein standard is spotted on the array (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more protein standardsare spotted on the array). Readout from the internal protein standardseries allows for quantification of proteins of interest in parallelfrom the biological sample (e.g., a tissue section) directly on thearray which can lead to increased accuracy when determining proteinconcentration. FIG. 26 shows an exemplary substrate with an exemplaryconfiguration that includes a dilution series for two different proteinsspotted on the margins of a spatial array. FIG. 26 shows a dilutionseries for two proteins of interest, however, as described herein morethan two protein dilution series can be spotted on the spatial array.

Provided herein are compositions such as spatial arrays including a) aplurality of capture probes including spatial barcodes and a firstplurality of capture domains hybridized to a plurality of templatedligation products, and b) a plurality of capture probes comprisingspatial barcodes and a second plurality of capture domains hybridized toa plurality of oligonucleotides from analyte capture agents, wherein theoligonucleotides comprise an analyte capture sequence and an analytebinding moiety barcode. In some compositions, the analyte capturesequences of the oligonucleotides are hybridized to the second pluralityof capture domains.

In some compositions, the capture probes include cleavage domains,unique molecular identifiers, functional sequences, or combinationsthereof. In some compositions, the first plurality of capture domainsare homopolymeric sequences. In some compositions, the first pluralityof capture domains comprise poly(T) sequences. In some compositions, thefirst plurality of capture domains are defined non-homopolymericsequences. In some compositions the first plurality of capture domainscomprise a degenerate sequence. In some compositions, the firstplurality of capture domains comprise a fixed sequence. For example, thefirst plurality of capture domains can comprise one of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

In some compositions, the second plurality of capture domains arehomopolymeric sequences. In some compositions, the second plurality ofcapture domains are defined non-homopolymeric sequences. In somecompositions, the second plurality of capture domains comprise poly(T)sequences. In some compositions the second plurality of capture domainscomprise a degenerate sequence. In some compositions, the secondplurality of capture domains comprise a fixed sequence. For example, thesecond plurality of capture domains can comprise one of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:11.

In some compositions, the spatial array comprises one or more proteindilution series.

Kits

Also provided herein are kits including a) a spatial array comprising aplurality of capture probes, where the capture probes include spatialbarcodes and wherein the plurality of capture probes comprise a firstplurality of first capture domains and a second plurality of secondcapture domains, b) one or more analyte capture agents, c) one or moreRNA templated ligation probe pairs, and d) one or more enzymes andbuffers for practicing any of the methods described herein. In somekits, one or more enzymes includes polymerases, RNases, DNases,proteases, lipases, or combinations thereof.

SEQUENCE LISTING SEQ ID NO: 1 - Capture Domain 22 merTTGCTAGGACCGGCCTTAAAGC SEQ ID NO: 2 - Capture Domain 21 merTTGCTAGGACCGGCCTTAAAG SEQ ID NO: 3 - Capture Domain 20 merTTGCTAGGACCGGCCTTAAA SEQ ID NO: 4 - Capture Domain 19 merTTGCTAGGACCGGCCTTAA SEQ ID NO: 5 - Capture Domain 18 merTTGCTAGGACCGGCCTTA SEQ ID NO: 6 - Capture Domain 17 merTTGCTAGGACCGGCCTT SEQ ID NO: 7 - Capture Domain 16 mer TTGCTAGGACCGGCCTSEQ ID NO: 8 - Capture Domain 15 mer TTGCTAGGACCGGCCSEQ ID NO: 9 - Capture Domain 14 mer TTGCTAGGACCGGCSEQ ID NO: 10 - Capture Domain 13 mer TTGCTAGGACCGGSEQ ID NO: 11 - Capture Domain 12 mer TTGCTAGGACCG

EXAMPLES Example 1. Spatial Proteomics and Gene Expression on FFPE HumanLymph Nodes

Experiments were undertaken to determine whether analyte capture agentscould provide for protein expression analysis concurrently withassociated gene expression. In these experiments, the analyte captureagent includes an antibody as the analyte binding moiety and the analytecapture sequence that comprises a barcode sequence that identifies theantibody as well as the capture sequence that is complementary to theassociated capture probe capture domain on the array.

Templated ligation probes were allowed to hybridize to their targetmRNAs and antibody-oligonucleotide conjugates were incubated with thesamples wherein the antibodies were allowed to bind to their proteintargets (as described in PCT/US2020/66720). Briefly, FFPE human lymphnodes tissues were sectioned, mounted on spatial array slides anddeparaffinized using a series of xylene and ethanol washes prior tobrightfield imaging. Tissues were washed and decrosslinked by incubatingthe tissues in TE (pH 9.0) buffer for 1 hr at 70° C. Afterdecrosslinking the tissues, the targeted templated ligation probes wereadded to the tissues and probe hybridization ran overnight at 50° C.Post hybridization, the probes were ligated together at 37° C. for 1 hr.Following templated ligation probe hybridization the analyte captureagents were added to the tissues, which were incubated in an antibodystaining buffer with the tissues overnight at room temperature. Thetissue samples were washed four times with the antibody staining bufferwithout antibodies.

Tissues were permeabilized and the ligated probe products, or ligationproducts, were allowed to migrate for hybridization to the capturedomains of the capture probes on the spatial array surface. Theoligonucleotides of the analyte capture agents also migrated in paralleland were captured via their capture sequences that hybridized to captureprobe capture domains on the spatial array that are complementary to thecapture sequences. As such, the ligation products representing thetarget mRNAs and the oligonucleotides of the analyte capture agentsrepresenting the binding of the antibodies to the targeted proteins wereconcurrently captured on the array surface. To allow for probe andoligonucleotide release and capture, the tissues were incubated withRNAse H and an associated buffer for 30 min at 37° C., tissuespermeabilized using a protease for an additional 40 min., followed bywashing to remove the enzymes from the tissues.

The captured ligation products and the analyte binding agentoligonucleotides were extended to create extended products of thecaptured molecules including the spatial barcode or a complementthereof, the analyte binding moiety barcode if present and otherfunctional sequences from the capture probe. Library preparations weremade, the libraries sequenced on an Illumina sequencing instrument, andspatial locations determined using Space Ranger and Loupe Browser (10XGenomics). The antibody sequences (e.g., the complement of the capturedoligonucleotide from the analyte binding agents) were amplified withTruseq_pR1 and Truseq_pR2. For protein localization, sequences relatingto the analyte binding moiety barcode were used to determine abundanceand location of the labeled protein by the analyte binding agents.Spatial expression patterns were determined using SpaceRanger dataanalysis software and Loupe browser visualization software (10XGenomics).

FIGS. 4A-B demonstrates the results of an experiment where analytecapture agents were combined to determine whether multiple targets couldbe identified concurrently in a tissue. In this experiment, eightdifferent antibodies were conjugated to oligonucleotides comprisinganalyte binding moiety barcodes and capture sequences targeting eightproteins: NCAM1, Ki67, CD8A, PDL1, CD20, CD11B, CD45RA and CD66B. FIG.4A (RNA) shows the spatial gene expression clustering of the associatedprotein in the lymph node tissue sample, whereas FIG. 4B (protein) showsthe spatial protein expression clustering of the eight targets. Thisexperiment demonstrates that it is feasible to multiplex differentanalyte capture agents to concurrently identify the spatial gene andprotein expression patterns of multiplex targets in a tissue sample.

Additional experimental results from the multiplexed experiment shown inFIG. 4A included targeting CD20 and CD20 mRNA when only one protein istargeted with an analyte capture agent. These results (data not shown)show a lymph node tissue sample where an antibody directed to CD20 wasconjugated to an oligonucleotide for subsequent capture on a spatialarray. mRNA from the tissue section was successfully captured andextended thereby providing CD20 spatial gene expression analysis (datanot shown). Concurrently, spatially presented protein array demonstratesthat the CD20 antibody of the analyte capture agent was able to bind tothe CD20 protein followed by oligonucleotide capture on the spatialarray, extension and spatial determination of the location of the CD20protein via the analyte binding moiety barcode (data not shown). Geneexpression and protein expression patterns for CD20 overlap and werelocalized to B-cells in the lymph nodes (data not shown). As such, themethods demonstrate the utility of the analyte capture agents to targeta protein of interest and simultaneously detect spatial gene expressionand protein expression in a tissue sample.

Additional data was generated with the target CD8A (e.g., using a CD8Aantibody) similar to CD20 described above. The spatial gene expressionand protein expression patterns were consistent with the targeting ofcytotoxic CD8 positive T cells in the lymph node tissue (data notshown), again demonstrating the utility of the methods and analytebinding agents that target specific proteins of interest forsimultaneously determining spatial gene and protein expression of agiven target.

Example 2. Spatial Proteomics and Gene Expression on FFPE Human TonsilTissue

As described above experiments were undertaken to determine whetheranalyte capture agents could provide for protein expression analysisconcurrently with associated gene expression in FFPE human tonsiltissue.

Briefly, FFPE human tonsil tissues were sectioned, mounted on spatialarray slides and deparaffinized using a series of xylene and ethanolwashes prior to H&E staining and brightfield imaging. Tissues werewashed and decrosslinked by incubating the tissues in citrate buffer (pH6.0) for 1 hour at 90° C. After decrosslinking the tissues, the targetedligation probes were applied to the tissues and probe hybridization ranovernight at 50° C. Post hybridization, the probes were ligated togetherat 37° C. for 1 hour to generate ligation products. Following ligationprobe hybridization the analyte capture agents were applied to thetissues, which were incubated in an antibody staining buffer (PBS-basedbuffer, 5% goat serum, salmon sperm DNA and dextran sulfate) with thetissues overnight at 4° C. The tissue samples were washed four timeswith antibody-free antibody staining buffer.

Tissues were permeabilized and the ligation products were allowed tomigrate for capture by hybridization to the capture domains of thecapture probes on the spatial array surface. The oligonucleotides of theanalyte capture agents complementary to the alternative capturesequences of the second set of capture probes on the array were alsocaptured by hybridization. As such, both ligation products representingthe mRNA of the targeted protein and the oligonucleotide of the analytecapture agent representing the binding of the antibody to the targetedprotein were concurrently captured on the array surface. To allow forprobe and oligonucleotide release and capture, the tissues wereincubated with RNase H and an associated buffer, and polyethylene glycol(PEG) for 30 minutes at 37° C. Tissues were permeabilized using apermeabilization buffer comprising a protease (e.g., Proteinase K), PEG,1M urea, for an additional 60 minutes, followed by washing to remove theenzymes from the tissues.

The captured ligation products and the analyte binding agentoligonucleotides were extended to include the spatial barcode or acomplement thereof, the analyte binding moiety barcode or a complementthereof if present and other functional sequences from the captureprobe. Additionally, said products were pre-amplified prior to librarypreparation.

Library preparations were made from the extended products, the librariessequenced on an Illumina sequencing instrument, and spatial locationsdetermined and visualized. The antibody sequences (e.g., the complementof the captured oligonucleotide from the analyte binding agents) wereamplified with Truseq_pR1 and Truseq_pR2. For protein localization,sequences relating to the analyte binding moiety barcode were used todetermine abundance and location of the labeled protein by the analytebinding agents. Spatial expression patterns were determined usingSpaceRanger data analysis software and Loupe browser visualizationsoftware (10X Genomics).

FIG. 5 shows exemplary clustering data of spatial gene (top middle) andprotein expression (top right) in FFPE tonsil tissue sectionssuperimposed on the H&E image (top left). FIG. 5 (bottom) showsvisualization of PD-1, Ki67, and CD8A protein markers labeling thefollicular T cells, individual follicles, and suppressor/cytotoxic Tcells, respectively. The data demonstrate the utility of analyte captureagents to target a protein of interest and simultaneously detect spatialgene expression in FFPE tonsil tissue samples. In addition, the datademonstrate the specificity of the protein markers (e.g., antibodies) toidentify different cell types within a heterogeneous tissue section andsimultaneously detect spatial gene expression.

In another experiment performed on FFPE tonsil tissue sections exemplaryspatial gene and protein expression for a 20-plexantibody-oligonucleotide conjugate targeting proteins of interest andRNA templated probes targeting 18,000 mRNA targets was performed. TheFFPE human tonsil tissue section was H&E stained which identified wherefollicles containing maturing immune cells and the epithelial layer canbe seen (data not shown). Representative gene expression cluster dataand representative protein expression cluster data align with themacroscopic structures visible in the H&E image shown (data not shown).

The data demonstrate the feasibility of multiplexing different analytecapture agents to concurrently identify the spatial gene and proteinexpression patterns of multiplex targets in a tissue sample.

FIG. 6 shows an exemplary H&E stained FFPE tonsil tissue section andspatial and gene expression data (top). FIG. 6 also shows an expandedview of the H&E stained FFPE tonsil tissue section and clustering ofgene expression and protein expression of CD8a and CD4 superimposed onthe H&E FFPE tonsil tissue section (bottom). Visualization of the CD8aand CD4 markers coincide with the follicles shown in the H&E stainingwhere CD8a localizes to the edge of the follicles and CD4 localizesoutside the follicles.

Additional exemplary clustering data of spatial gene and proteinexpression was performed on a different FFPE tonsil tissue sections.Spatial gene expression clustering, spatial protein clustering wasperformed on the FFPE tonsil tissue sample which was also H&E stained(data not shown). The results demonstrate an experiment where analytecapture agents were combined to determine whether multiple targets couldbe identified concurrently in FFPE tonsil tissue. In this experiment 21different antibodies were conjugated to oligonucleotides comprisinganalyte binding moiety barcodes and capture sequences (e.g., analytecapture sequences) targeting 21 proteins (18 shown in FIG. 7 ): Ki67,NCAM1, BCL, CD138, CD21, CD268, CD11b, LAG3, CD4, CD20, PD-L1, CD45RA,CD68, PanCK, CD66b, PCNA, CD235a/b and CD79a.

FIG. 7 shows a panel of 18 of the 21 targeted proteins includingspecifically genes: Ki67, NCAM1, BCL, CD138, CD21, CD268, CD11b, LAG3,CD4, CD20, PD-L1, CD45RA, CD68, PanCK, CD66b, PCNA, CD235a/b and CD79a.In the panel the top row shows protein expression and the bottom panelshows RNA expression. Expanded views of exemplary spatial gene andprotein expression from a subset of seven different targeted gene andprotein expression heat maps, including specifically: PanCK, PD-L1,Ki67, CD4, CD68, CD20, and CD11b. Gene expression (RNA) and proteinexpression for Ki67, NCAM1, BCI, CD138, CD11b, LAG, CD4, CD20, PD-L1,PanCK, CD66b, and CD79a as shown in FIG. 7 overlap, demonstrating theutility of the analyte capture agents to target a protein of interestand simultaneously detect spatial gene expression in FFPE tonsil tissuesamples (data not shown).

FIG. 8 shows expanded views of exemplary clustering data for FFPE humantonsil tissue using a 12-plex antibody-oligonucleotide conjugate testpanel of nine genes shown in FIG. 7 , including specifically: CD4,PD-L1, CD20, CD11b, BCL-2, CD21, CD791, CD45RA, PCNA, Ki67, PanCK, andCD235a/b.

Collectively, the data demonstrate the utility of the methods andanalyte binding agents that target specific proteins of interest forsimultaneously determining spatial gene and protein expression of agiven target.

Example 3. Spatial Proteomics and Gene Expression on FFPE Breast CancerTissue Human Triple Positive Breast Cancer Tissue

As described above experiments were undertaken to determine whetheranalyte capture agents could provide for protein expression analysisconcurrently with associated gene expression in FFPE human triplepositive breast cancer tissue. Briefly, FFPE human triple positivebreast cancer tissues were sectioned, mounted on spatial array slidesand deparaffinized using a series of xylene and ethanol washes prior todrying at room temperature. Next, the slides were heated at 37° C. for15 minutes, followed by a series of ethanol washes (100%, 96%, 96%, and70% ethanol). Next, the tissues were H&E stained and brightfield imaged.Alternatively, tissues can be stained (e.g., immunofluorescence stained)instead of H&E staining.

Tissues were washed and decrosslinked by incubating the tissues inTris-EDTA (TE) buffer (pH 9.0) for 1 hour at 95° C. followed by a seriesof washes with 0.1 N HCl. After decrosslinking the tissues, the targetedligation probes were added to the tissues and probe hybridization ranovernight at 50° C. The tissues were washed in a post-hybridizationbuffer including 3×SSC, Baker's yeast tRNA, and nuclease free water andfollowed by a 2×SSC buffer wash. Post-hybridization, the probes wereligated together at 37° C. for 1 hour. Following probe hybridization,the tissues were incubated in an antibody blocking buffer (PBS-basedbuffer (pH 7.4), goat serum, salmon sperm DNA, Tween-20, an RNaseinhibitor, and dextran sulfate) at room temperature for 60 minutes. Theblocking buffer was removed from the tissues and the tissues wereincubated overnight at 4° C. with the analyte capture agents in anantibody staining mixture (PBS-based buffer (pH 7.4), 5% goat serum, 0.1μg/μL salmon sperm DNA, 0.1% Tween-20, 1 U/μL RNase inhibitor, blockingoligonucleotides, analyte capture agents (e.g., antibodies with aconjugated oligonucleotide) and 10 mg/mL dextran sulfate)). The tissuesamples were washed several times with antibody staining buffer withoutantibodies.

Tissues were permeabilized and the ligated probes were released forcapture by hybridization to the capture domains of the capture probes onthe spatial array surface. The oligonucleotides of the analyte captureagents complementary to the alternative capture sequences of the secondset of capture probes on the array were also captured by hybridization.As such, both ligation products representing the target mRNA and theoligonucleotide of the analyte capture agent representing the binding ofthe antibody to the targeted protein were concurrently captured on thearray surface. To allow for probe and oligonucleotide release andcapture, the tissues were incubated with an RNase (e.g., RNase H), anassociated buffer, and polyethylene glycol (PEG) for 30 minutes at 37°C. Tissues were permeabilized using a permeabilization buffer comprisinga protease (e.g., Proteinase K), PEG, 3M urea, for an additional 60minutes, followed by washing to remove the enzymes from the tissues.After permeabilization the tissues were washed in 2×SSC several times.

The captured ligation products and the analyte binding agentoligonucleotides were extended to create extended products of thecaptured molecules including the complement of the spatial barcode, theanalyte binding moiety barcode if present and other functional sequencesfrom the capture probe. Additionally, said products were pre-amplifiedprior to library preparation.

Library preparations were made from the extended products, sequenced onan Illumina sequencing instrument, and spatial locations were determinedand visualized. The antibody sequences (e.g., the complement of thecaptured oligonucleotide from the analyte binding agents) were amplifiedwith Truseq_pR1 and Truseq_pR2. For protein localization, sequencesrelating to the analyte binding moiety barcode were used to determineabundance and location of the labeled protein by the analyte bindingagents. Spatial expression patterns were determined using SpaceRangerdata analysis software and Loupe browser visualization software (10XGenomics).

FIGS. 9A-C demonstrate the results of an experiment where analytecapture agents were combined to determine whether targets could beidentified concurrently in grade II

FFPE triple positive invasive ductal carcinoma breast cancer tissue. Inthis experiment 11 different antibodies were conjugated tooligonucleotides comprising analyte binding moiety barcodes and capturesequences (e.g., analyte capture sequences) targeting eleven proteins:Her2, EpCAM, PanCK, N-Cadherin, PCNA, AlphaSMA, Vimentin, CD8a, CD4,CD68, CD20, and HLA-DR. FIG. 9B (RNA) shows the spatial gene expressionclustering, whereas FIG. 9C (protein) shows clustering of the associatedprotein in the FFPE triple positive breast cancer tissue sample. Thedata demonstrate the feasibility of multiplexing different analytecapture agents to concurrently identify the spatial gene and proteinexpression clusters patterns of multiplex targets in triple positivebreast cancer tissue samples.

A panel of 11 targeted proteins including specifically genes: Her2,EpCAM, PanCK, N-Cadherin, PCNA, AlphaSMA, Vimentin, CD8a, CD4, CD68,CD20, and HLA-DR was generated where for each target RNA expression andprotein expression was generated (data not shown). Exemplary spatialgene and protein expression for KRT10, KRT18, and PanCK Ab was alsogenerated and unbiased clustering of gene and protein expressionsuperimposed on the H&E image demonstrate similar patterns (data notshown). An expanded view of Her2 and Vimentin RNA expression and proteinexpression is shown in FIG. 10 . Adjacent sections wereimmunofluorescently stained for HER2 (top) and Vimentin (bottom)demonstrating strong signal within the dashed box region of the biopsysample. Antibody staining for two biomarkers, HER2 and Vimentin, fromadjacent tissue sections correlate with tumors in triple positive breastcancer tissue. Gene expression (RNA) and protein expression for Her2,EpCAM, PanCK, N-Cadherin, PCNA, AlphaSMA, Vimentin, CD8a, CD4, CD68,CD20, and HLA-DR also overlap (data not shown) demonstrating the utilityof the analyte capture agents to target a protein of interest andsimultaneously detect spatial gene expression in triple positive breastcancer tissue samples.

As described above experiments were undertaken to determine whetheranalyte capture agents could provide for protein expression analysisconcurrently with associated gene expression in FFPE invasive ductalcarcinoma breast cancer tissue.

The samples were prepared as described above in the FFPE triple positivebreast cancer example. FIGS. 11A-C show H&E-stained human invasiveductal carcinoma tissue (FIG. 11A). FIGS. 11B and 11C show geneexpression clustering and protein expression clustering, respectively,superimposed on the H&E image with similar patterns. FIG. 12 showsadditional examination of different biomarkers: CD11b, PD-L1, andalpha-SMA exhibit regional variation within the tissue sample. Theseprotein biomarkers were used to define the region of interest (e.g.,shown as region 1 and region 2). The identified regions of interestspecific gene expression patterns were identified with the top 50 highlyexpressed genes. The data demonstrate the feasibility of multiplexingdifferent analyte capture agents to concurrently identify the spatialgene and protein expression cluster patterns of multiplex targets ininvasive ductal carcinoma tissue samples.

FIG. 13 shows exemplary spatial gene and protein expression for Acta2(top) and Epcam (bottom). Unbiased clustering of gene and proteinexpression superimposed on an H&E image of the same tissue sampledemonstrate similar patterns. Plots showing the correlation betweenActa2 gene expression and Alpha-SMA protein expression (encoded by theActa2 gene) and Epcam gene expression and EPCAM protein expression(encoded by the EPCAM gene) were also generated (data not shown). Asimilar experiment was performed as described in FIG. 13 on a differentFFPE invasive ductal carcinoma breast cancer tissue and the exemplaryspatial gene and protein expression for Acta2 and separately HLA-DRA(gene) and HLA-DR (protein) correlated with one another (data notshown).

Collectively, the data demonstrate the feasibility of multiplexingdifferent analyte capture agents to concurrently identify the spatialgene and protein expression in triple positive FFPE invasive ductalcarcinoma tissue samples.

Example 4. Spatial Proteomics and Gene Expression on FFPE Mouse Spleen,Brain, Head, and Torso Sections in a Mouse Embryo Sample

Experiments were undertaken to determine whether analyte capture agentscould provide for protein expression analysis concurrently withassociated gene expression in FFPE mouse tissues, including whole mouseembryos or portions thereof. The following experiments tested spatialgene and protein expression in sandwiching and non-sandwiching formats.In some examples, the alignment of a first substrate with a biologicalsample and a second substrate with a spatial array thereon isfacilitated by a sandwiching process. Accordingly, described herein aremethods of sandwiching together the first substrate with a biologicalsample with a second substrate comprising an array with a plurality ofcapture probes, where the capture probe includes a spatial barcode and acapture domain.

In a non-limiting example, FFPE mouse spleen samples, FFPE mousesamples, FFPE mouse embryo torso samples, FFPE mouse embryo head sampleswere placed onto standard slides (for sandwich conditions) or spatialexpression (GEx) slides (as non-sandwich conditions). GEx slides includean array of spatially barcoded capture probes. Briefly, tissues weresectioned and mounted on slides and dried overnight in a desiccator. Thefollowing day, the tissues were heated to 60° C., followed bydeparaffinization and rehydration. Tissues were H&E stained andbright-field imaged. Tissues were destained using HCl and decrosslinkedfor 1 hour in citrate buffer (pH 6.0) at 95° C. After decrosslinking,tissues were incubated overnight with whole mouse transcriptome (RNAtemplated ligation) probe sets at 50° C. The following day, tissues werewashed to remove un-hybridized probes, then treated with ligase toligate together the RTL probes. After another wash step, the tissueswere blocked with antibody blocking buffer. Tissues were incubatedovernight with a library of conjugated antibodies (e.g., a librarycomprising a plurality of analyte capture agents, each comprising anantigen specific antibody conjugated to an oligonucleotide). Thefollowing day, tissues were subjected to sandwiching or non-sandwichconditions as follows.

Tissues placed on standard slides for the sandwiching conditions werewashed with PBS-T, subjected to an eosin stain, and washed with SSC. Thetissues were subjected to sandwiching conditions. Briefly, the tissueslides were mounted in a sandwiching instrument along with a GEx slideand a reagent solution including an RNAse and Proteinase K. Upon closurein the instrument, the tissue sections were permeabilized for 30 min.allowing the ligation products and the oligonucleotides from the analytecapture agents to migrate to the GEx slide for capture by the captureprobes. Following permeabilization and capture, the GEx slides wereremoved from the instrument.

Tissues placed on GEx slides for non-sandwiching conditions were washedwith PBS-T and SSC. The tissues were subjected to a 30 min probe releasestep with an RNase, followed by permeabilization with a permeabilizationbuffer including Proteinase K. Accordingly, the ligation products andanalyte capture agents were captured by the capture probes on the GExslide.

Regardless of conditions, GEx slides were washed twice with 2×SSC, andsubjected to probe extension, denaturation, and pre-amplificationfollowed by amplification and sequencing of the templated ligation andanalyte capture agent libraries.

After sequencing, the quality, sensitivity, and detection under eachcondition (sandwiching and non-sandwiching conditions) was evaluated. Asshown in Table 1, the quality, sensitivity, and detection ofglobally-detected transcripts (i.e., mRNA) and proteins were comparableacross the sandwich and non-sandwich conditions.

TABLE 1 Sandwiching Non-Sandwiching Metric Conditions ConditionsTemplated Valid barcodes 99.00% 98.90% Ligation Fraction reads 85.60%82.10% Quality on target Fraction reads 79.80% 78.70% usable Fractionreads 81.60% 81.00% in spots Fraction reads 0.90% 1.00% unmappedTemplated Median genes 4856 4705 Ligation (20K prps) Sensitivity MedianUMIs 16966 15156 (20K prps) Protein Fraction reads 75.20% 67.10%Detection usable using Fraction reads 78.70% 70.10% Analyte in spotCapture Fraction unknown 3.70% 3.60% Agents Median UMIs per 4632 4114spot (5K reads usable per spot) Correlation of 0.77 0.76 selectedTemplated Ligation/Analyte Capture Agent

Images were generated to evaluate the overlap of gene expression andgene protein profiles in mouse spleen tissue and mouse brain tissue forindividual biomarkers. As shown in FIG. 14A (sandwiching conditions) andFIG. 14B (non-sandwiching conditions), tyrosine phosphatase receptortype C (Ptprc; e.g., Ensembl: ENSMUSG00000026395) mRNA expression wasdetected in a spleen tissue sample. Further, gene product (e.g.,protein) CD45R was also detected both in sandwiching conditions (FIG.14C) and in non-sandwiching conditions (FIG. 14D). CD45R is the proteinname of Ptprc, and it was determined whether there was overlap of mRNAexpression of Ptprc and protein expression of CD45R. As shown in FIGS.14A-14D, both sandwiching (77% correlation) and non-sandwichingconditions (76% correlation), respectively, demonstrates overlap oftranscript and protein, indicating that transcript (i.e., mRNA) andprotein detection (1) was identified in similar areas of the tissues and(2) was comparable across the sandwich and non-sandwich conditions inmouse spleen samples.

The quality, sensitivity, and detection under each condition(sandwiching and non-sandwiching conditions) were evaluated globally inmouse brain samples. As shown in Table 2, the quality, sensitivity, anddetection of globally-detected transcripts (i.e., mRNA) and proteinswere comparable across the sandwich and non-sandwich conditions in mousebrain samples.

TABLE 2 Sandwiching Non-Sandwiching Metric Conditions ConditionsTemplated Valid barcodes 99.00% 98.90% Ligation Fraction reads 92.00%87.30% Quality on target Fraction reads 88.80% 72.30% usable Fractionreads 91.80% 79.10% in spots Fraction reads 1.10% 1.90% unmappedTemplated Median genes 4405 3185 Ligation (10K prps) Sensitivity MedianUMIs 9386 5553 (10K prps) Protein Fraction reads 80.70% 62.30% Detectionusable using Fraction reads 84.40% 65.10% Analyte in spot CaptureFraction unknown 3.70% 3.70% Agents Median UMIs per 3232 3221 spot (5Kreads usable per spot) Correlation of 0.63 0.63 selected TemplatedLigation/Analyte Capture Agent

Similar to the mouse spleen sample images described above, individualgene expression and protein products were evaluated in mouse brainsamples. As shown in FIG. 15A (sandwiching conditions) and FIG. 15B(non-sandwiching conditions), tyrosine hydroxylase (Th; e.g., Ensembl:ENSMUSG00000000214) mRNA expression was detected in brain. Further, thegene product (e.g., protein) TH was also detected in sandwichingconditions (FIG. 15C) and non-sandwiching conditions (FIG. 15D). SinceTH is the protein made by Th, it was determined whether there wasoverlap of mRNA expression of Th and protein expression of TH. As shownin FIGS. 15A-15D, both sandwiching (63% correlation) and non-sandwichingconditions (63% correlation), respectively, saw overlap of transcriptand protein, demonstrating that transcript (i.e., mRNA) and proteindetection was comparable across the sandwich and non-sandwich conditionsin mouse brain samples.

Experiments using the same methods (i.e., testing sandwiching conditionsversus non-sandwiching conditions while detecting both RNA and protein)were performed on whole mouse embryo torso and head sections. Inaddition to conditions in which both RNA and protein were detected, athird condition was included as a control. This third condition(Condition 3 in Tables 3 and 4) detected the presence and abundance ofonly RNA. In each condition, RNA was detected using templated ligationas previously described. For Conditions 1 and 2 as shown in Tables 3 and4 below, protein was also detected using analyte capture agent methodsas previously described. The quality, sensitivity, and detection undereach condition (sandwiching versus non-sandwiching conditions) wereevaluated in mouse embryo torso and head samples. As shown in Tables 3and 4, the quality, sensitivity, and detection of globally-detectedtranscripts (i.e., mRNA) and proteins were comparable across thesandwich and non-sandwich conditions in mouse embryo torso andhead/upper torso samples. Further, the quality, sensitivity, anddetection of globally-detected transcripts (i.e., mRNA) was roughly thesame between Conditions 1 and 3, demonstrating that both protein captureand sandwiching methods did not interfere with RNA capture usingtemplated ligation methods.

TABLE 3 Whole Mouse Embryo Torso Sample Data Condition 2: Condition 1:Non- Sandwiching Sandwiching Condition 3: Conditions Conditions Non-Detecting Detecting Sandwiching both RNA and both RNA and ConditionsMetric Protein Protein Detecting RNA Templated Valid barcodes 98.9%99.0% 98.5% Ligation Fraction reads 88.5% 88.5% 87.9% Quality on targetFraction reads 81.5% 77.7% 84.8% usable Fraction reads 84.0% 79.9% 88.4%in spots Fraction reads 1.0% 1.0% 1.4% unmapped Templated Median genes4144 4356 3562 Ligation (10K prps) Sensitivity Median UMIs 8767 81766121 (10K prps) Protein Fraction reads 77.3% 72.6% — Detection usableusing Fraction reads 80.8% 75.9% — Analyte in spot Capture Fractionunknown 3.7% 3.7% — Agents Median UMIs per 3358 3264 — spot (5K readsusable per spot) Correlation of 0.90 0.80 — selected TemplatedLigation/Analyte Capture Agent

TABLE 4 Whole Mouse Embryo Head/Upper Torso Sample Data Condition 2:Condition 1: Non- Condition 3: Sandwiching Sandwiching Non- ConditionsConditions Sandwiching Detecting Detecting Conditions both RNA and bothRNA and Detecting Metric Protein Protein RNA Templated Valid barcodes99.0% 99.0% 98.5% Ligation Fraction reads 89.1% 89.4% 89.0% Quality ontarget Fraction reads 86.7% 82.9% 82.2% usable Fraction reads 89.5%85.2% 85.2% in spots Fraction reads 1.0% 1.0% 1.5% unmapped TemplatedMedian genes 4400 4708 3562 Ligation (10K prps) Sensitivity Median UMIs9077 8431 6571 (10K prps) Protein Fraction reads 84.3% 77.9% — Detectionusable using Fraction reads 88.2% 81.5% — Analyte in spot CaptureFraction unknown 3.7% 3.8% — Agents Median UMIs per 3358 3349 — spot (5Kreads usable per spot) Correlation of 0.72 0.59 — selected TemplatedLigation/Analyte Capture Agent

In addition to performing global expression analysis on each group,individual targets were analyzed for the location and abundance ofspatial gene expression (e.g., mRNA) and spatial protein expression ofsingle targets in the mouse embryo torso and head/upper torso samples.FIGS. 16A and 16C show mRNA (FIG. 16A) and protein (FIG. 16C) detectionof lymphocyte antigen 76 (Ter119) (e.g., NCBI Gene ID: 104231), of mouseembryo torso samples in Condition 1 (i.e., in sandwiching conditions).FIGS. 16B and 16D show mRNA (FIG. 16B) and protein (FIG. 16D) detectionof Ter119 of mouse embryo torso samples in Condition 2 (i.e., innon-sandwiching conditions). FIGS. 17A and 17C show mRNA (FIG. 17A) andprotein (FIG. 17C) detection of Ter119 of mouse embryo head samples inCondition 1 (i.e., in sandwiching conditions). FIGS. 17B and 17D showmRNA (FIG. 17B) and protein (FIG. 17D) detection of Ter119 of mouseembryo head samples in Condition 2 (i.e., in non-sandwichingconditions). As shown in FIGS. 17A-17D, Ter119 mRNA and protein wasreadily detected with considerable overlap of mRNA and protein detectionin both sandwiching conditions and non-sandwiching conditions,demonstrating the adaptability and reproducibility of the methodsregardless of condition.

Additional single biomarkers were analyzed in the mouse embryo torso andhead samples. As shown in FIGS. 18A-18I, metallophosphoesterase domaincontaining 1 (Mpped1; Ensemb1: ENSMUSG00000041708) mRNA and protein wasanalyzed. Mpped1 was readily detected in the brain region of mouseembryo head samples. FIG. 18A shows detection of Mpped1 RNA in a mouseembryo head sample using sandwiching conditions (Condition 1 of Tables 3and 4). FIG. 18E shows detection of Mpped1 protein in a mouse embryohead sample using sandwiching conditions (Condition 1 of Tables 3 and4). However, Mpped1 RNA was not readily detected in a mouse embryo torsosample (FIG. 18B). Consistent with these observations, Mpped1 hasmetallophosphoesterase activity, which could have a role in braindevelopment, as such was expected to be present in the embryo headsample and not in the embryo torso sample. Consistent with thesandwiching conditions data, Mpped1 mRNA and protein was detected innon-sandwiching conditions in the head (FIG. 18C (mRNA) and FIG. 18F(protein)) but not in the torso (FIG. 18D (mRNA) and FIG. 18G(protein)). Similarly, in non-sandwiching conditions in which only RNAwas detected, Mppedl RNA was detected in the head (FIG. 18H) but not inthe torso (FIG. 18I). As such, regardless of conditions, both gene andprotein expression, down to the single biomarker level, can be detectedconcurrently in the same sample.

Four additional biomarkers—troponin C1, slow skeletal and cardiac type(Tnnc1; e.g., Ensembl: ENSMUSG00000091898); fibroblast growth factor 15(Fgf15; e.g., Ensemb1: ENSMUSG00000031073); epiphycan (Epyc e.g.,Ensembl: ENSMUSG00000019936); and serine (or cysteine) peptidaseinhibitor, Glade A, member 1 E (Serpinale; e.g., Ensembl:ENSMUSG00000072849) were examined in head/upper torso and torso mouseembryo samples under Conditions 1, 2, and 3 from Tables 3 and 4. SeeFIGS. 19A-22I. Tnnc1 is involved in muscle contraction regulation andits expression was readily detected in all samples under each Condition.See FIGS. 19A-19I. Fgf15 functions in retinal neurogenesis and as a cellfate determination factor. Indeed, its expression was found in the eyeof the embryo in each head sample, but was not detected in the torso.See FIGS. 20A-20I. Epyc functions in bone formation and in cartilagestructure and was detected in each sample (head and torso). See FIGS.21A-21I. Finally, Serpinale is active in the liver as it functions inalpha-1 antitrypsin protein production. As shown in FIGS. 22A-22I,Serpinale was detected in the torso of mouse embryos but was notdetected in the head/upper torso samples. Consistent among each of thesebiomarker images, the non-sandwiching methods readily detectedindividual mRNA and protein biomarkers compared to sandwiching methods.

As such, using sandwiching or non-sandwiching conditions, both gene andprotein expression, down to the single biomarker level, can be detectedconcurrently across multiple tissue types using the methods describedherein.

Example 5. Spatial Proteomics, Spatial Gene Expression, and Immune CellIdentification in FFPE Cancer Tissue Sections

Experiments were undertaken to determine whether analyte capture agentscould provide for spatial protein and gene expression analysis in FFPEcancer tissue sections. Additionally, experiments were undertaken toidentify various types of immune cells in breast cancer FFPE tissuesections and ovarian cancer FFPE tissue sections. The tissue sectionswere prepared and analysis was performed by the methods described inExample 4.

Invasive Ductal Carcinoma Breast Cancer

As shown in FIGS. 23A-E a 25-plex antibody panel was used to study thetumor microenvironment and show spatial immune cell infiltration in abreast cancer FFPE tissue section. The data shown in FIGS. 23A-E arefrom cored samples from larger tissue sections which demonstratedconsistent spatial gene and spatial protein expression as shown in FIGS.23C-E and described herein (data not shown). FIG. 23A shows an H&Estained human invasive ductal carcinoma grade III breast cancer FFPEtissue section and FIG. 23B shows the H&E image of FIG. 23A annotated bya pathologist. The pathologist annotations are outlined in the image andcorrespond to either Blood Vessel, DCIS (ductal carcinoma in situ),Immune Cells, Invasive Carcinoma, Necrosis, or Normal Gland. Thepathologist identified ductal carcinoma in situ as well as areas oftissue with immune cell infiltrates.

The spatial gene and spatial protein expression clustering superimposedon the H&E stained image shown in FIG. 23A demonstrated similarexpression patterns delineating the tumor and stromal region (data notshown). More specifically, protein immune markers were used to identifyinfiltrating immune cells. For example, FIG. 23C shows spatial proteinexpression of CD3 (e.g., a marker for T cells) and FIG. 23D showsspatial protein expression of CD8A (e.g., a marker for cytotoxic Tcells). Moreover, within an infiltrate, spatial orientation of differenttypes of immune cells were distinguishable. An example of this is shownin FIG. 23E where an accumulation of cytotoxic T cells expressing humanleukocyte antigen—DR isotype (HLA-DR) (e.g., a marker of T cellactivation) were observed.

Additionally, spatial gene and protein expression of additional genesand proteins were examined from the same tissue section shown in FIG.23A. More specifically, gene ACTA2 and its corresponding protein,alpha-smooth muscle actin (SMA), demonstrated similar expressionpatterns with positive gene-protein UMI count correlations (data notshown). Further examination of cytokeratin gene, KRT18, alsodemonstrated similar patterns to a PanCK antibody and again positivegene-protein UMI correlations were observed (data not shown).

In a different grade II invasive ductal carcinoma FFPE breast cancertissue section the tissue section was H&E stained and spatial proteinexpression of PanCK and HLA-DR within the tissue section. Manualselection of the HLA-DR and PanCK positive regions was performed withthe 10x Loupe Browser showing contrasting regions within the tissuesection. Local differential expression analysis of both regionsgenerated the top 50 genes associated with expression in the HLA-DR orPanCK selected regions identified and are shown in Table 5.

TABLE 5 HLA-DR PanCK Marker HLA-DR Log2 Fold HLA-DR PanCK Log2 FoldPanCK Marker ID Name Average Change P-Value Average Change P-ValueENSG00000106483 SFRP4 11.59067363 2.58736286 3.34E−49 1.926061057−2.58736286 3.34E−49 ENSG00000168685 IL7R 3.519297315 2.4330618741.02E−40 0.650696303 −2.433061874 1.02E−40 ENSG00000011465 DCN29.40019273 2.218895222 4.74E−39 6.307770284 −2.218895222 4.74E−39ENSG00000091986 CCDC80 3.432380045 2.310230989 7.36E−39 0.691066033−2.310230989 7.36E−39 ENSG00000139329 LUM 33.17683334 2.1782906729.94E−38 7.321262975 −2.178290672 9.94E−38 ENSG00000197614 MFAP53.117091908 2.247513206 2.12E−36 0.655476929 −2.247513206 2.12E−36ENSG00000211772 TRBC2 4.483908589 2.20449644 3.84E−36 0.971529419−2.20449644 3.84E−36 ENSG00000087245 MMP2 9.819947279 2.176664543.84E−36 2.169341797 −2.17666454 3.84E−36 ENSG00000182326 C1S7.999797384 2.155136429 4.02E−36 1.793797074 −2.155136429 4.02E−36ENSG00000108821 COL1A1 150.9804112 2.114722952 4.02E−36 34.81889197−2.114722952 4.02E−36 ENSG00000136235 GPNMB 5.620650143 2.1615897482.39E−34 1.254648708 −2.161589748 2.39E−34 ENSG00000090382 LYZ5.075286879 2.204681169 3.37E−34 1.099543957 −2.204681169 3.37E−34ENSG00000064205 CCN5 3.718695759 2.138373605 4.86E−34 0.843514881−2.138373605 4.86E−34 ENSG00000211751 TRBC1 4.436189304 2.1851299847.54E−34 0.974185322 −2.185129984 7.54E−34 ENSG00000164692 COL1A297.7921546 2.041795601 8.18E−34 23.72199689 −2.041795601 8.18E−34ENSG00000277734 TRAC 4.685011293 2.099070424 4.58E−33 1.092107428−2.099070424 4.58E−33 ENSG00000211592 IGKC 190.0999999 2.2031760069.62E−33 41.23342956 −2.203176006 9.62E−33 ENSG00000271503 CCL54.115788386 2.085695324 1.34E−32 0.968342335 −2.085695324 1.34E−32ENSG00000158747 NBL1 8.572428812 2.007423552 1.09E−31 2.129503248−2.007423552 1.09E−31 ENSG00000106624 AEBP1 24.52600855 1.9861087681.64E−31 6.183474011 −1.986108768 1.64E−31 ENSG00000169442 CD522.54616474 2.103282373 1.85E−31 0.59173525 −2.103282373 1.85E−31ENSG00000168542 COL3A1 55.19587513 1.945997239 8.85E−31 14.30841332−1.945997239 8.85E−31 ENSG00000163520 FBLN2 4.983256828 1.9941159038.85E−31 1.249336902 −1.994115903 8.85E−31 ENSG00000172724 CCL194.214635477 2.569106092 1.13E−30 0.709126175 −2.569106092 1.13E−30ENSG00000145423 SFRP2 12.30135013 1.975672819 1.40E−30 3.123873435−1.975672819 1.40E−30 ENSG00000140937 CDH11 6.69433407 1.9735683411.57E−30 1.702434001 −1.973568341 1.57E−30 ENSG00000106565 TMEM176B2.784761169 2.020982234 4.69E−30 0.685223046 −2.020982234 4.69E−30ENSG00000172061 LRRC15 6.252930678 1.96380266 5.20E−30 1.600978496−1.96380266 5.20E−30 ENSG00000166741 NNMT 3.541452698 1.9790471111.90E−29 0.897164127 −1.979047111 1.90E−29 ENSG00000184347 SLIT32.425162266 2.012530816 3.18E−29 0.600234141 −2.012530816 3.18E−29ENSG00000107562 CXCL12 6.52390805 1.925167509 5.56E−29 1.715713517−1.925167509 5.56E−29 ENSG00000149131 SERPING1 7.503857666 1.9121044725.56E−29 1.991396278 −1.912104472 5.56E−29 ENSG00000123500 COL10A16.305762744 1.924239545 7.60E−29 1.659408368 −1.924239545 7.60E−29ENSG00000140285 FGF7 1.174235279 2.165189314 8.22E−29 0.261340883−2.165189314 8.22E−29 ENSG00000115594 IL1R1 2.104761348 2.0018169161.23E−28 0.524806488 −2.001816916 1.23E−28 ENSG00000159403 C1R10.59027289 1.895587338 2.40E−28 2.842878868 −1.895587338 2.40E−28ENSG00000165507 DEPP1 5.92741698 2.006365507 2.68E−28 1.473495138−2.006365507 2.68E−28 ENSG00000142871 CCN1 11.35378146 1.89404563.90E−28 3.051101685 −1.8940456 3.90E−28 ENSG00000143196 DPT 2.2121297411.984447325 3.90E−28 0.558270869 −1.984447325 3.90E−28 ENSG00000213886UBD 1.508270278 2.211600224 3.90E−28 0.325082561 −2.211600224 3.90E−28ENSG00000186340 THBS2 6.307467004 1.90036637 3.90E−28 1.687560943−1.90036637 3.90E−28 ENSG00000060718 COL11A1 5.138344506 1.9531990931.11E−27 1.325295736 −1.953199093 1.11E−27 ENSG00000197747 S100A106.643206264 1.86886665 1.72E−27 1.816637842 −1.86886665 1.72E−27ENSG00000132386 SERPINF1 11.63498439 1.853278452 1.80E−27 3.216298869−1.853278452 1.80E−27 ENSG00000118523 CCN2 16.70174997 1.8607430313.07E−27 4.593119128 −1.860743031 3.07E−27 ENSG00000127083 OMD1.624159972 2.000597227 4.93E−27 0.40529084 −2.000597227 4.93E−27ENSG00000084636 COL16A1 3.253432724 1.891287213 6.63E−27 0.875916901−1.891287213 6.63E−27 ENSG00000180447 GAS1 2.45413469 1.9015721912.20E−26 0.65600811 −1.901572191 2.20E−26 ENSG00000163430 FSTL110.87829286 1.80177238 6.32E−26 3.116436906 −1.80177238 6.32E−26ENSG00000227507 LTB 2.731929102 1.888847173 8.98E−26 0.736747569−1.888847173 8.98E−26

The data demonstrate that spatial protein expression can identify immunecell infiltration in breast cancer FFPE tissue sections and moreover thespatial protein expression correlates with annotations by a pathologistwhich demonstrates the utility of the methods described herein inidentifying immune cells within a tumor microenvironment can be used asa diagnostic tool.

Ovarian Carcinoma

As shown in FIGS. 24A-D a 25-plex antibody panel was used to study anFFPE ovarian cancer (e.g., carcinoma) tissue section and show spatialimmune cell infiltration. The antibody panel included antibodies forboth intracellular and extracellular markers. FIG. 24A shows apathologist's annotation for invasive carcinoma and immune cells in anH&E stained ovarian cancer FFPE tissue section. The pathologistannotations are outlined in the image and correspond to either BloodVessel, DCIS (ductal carcinoma in situ), Immune Cells, InvasiveCarcinoma, or Necrosis.

The 25-plex antibody panel included antibodies for protein immunemarkers which confirmed the presence of immune cells within the ovariancancer FFPE tissue section. Further, the antibody panel distinguished(e.g., subtyped) the immune cells based on their characteristic surfacemarkers. For example, FIG. 24B shows spatial protein expression of CD20(e.g., a marker for B cells) and FIG. 24C shows spatial proteinexpression of CD68 (e.g., a marker for monocytes). FIG. 24D showsspatial protein expression of CD8A (e.g., a marker for cytotoxic Tcells) and a small region of the ovarian carcinoma includes cytotoxic Tcell infiltration (arrow) while the larger carcinoma does not.

FIGS. 25A-D show differential spatial cytotoxic T cell infiltrationwithin different regions of an ovarian cancer FFPE tissue section. Geneexpression profiles of the large and small carcinoma regions werecompared to find differences that may be correlated to varying immunecell infiltration observed in the protein expression data. FIG. 25Ashows spatial protein expression of CD8A (e.g., a marker for cytotoxic Tcells) and FIG. 25B shows highly infiltrated (“hot”) and non-infiltrated(“cold”) areas of the ovarian cancer FFPE tissue section. Theinfiltration mapping is based on protein detection of CD3 proteinexpression for the “hot” or high cytotoxic T cell infiltration, and CD8protein expression for the “cold” or minimal to no cytotoxic T cellinfiltration. FIGS. 25C and 25D show spatial gene expression of immuneresponse genes. For example, FIG. 25C shows spatial gene expression ofHLA class I histocompatibility antigen, alpha chain G (HLA-G; also knownas human leukocyte antigen G) in the small carcinoma which correlateswith protein expression data and FIG.

shows spatial gene expression in the large carcinoma ofinterphotoreceptor matrix proteoglycan 2 (IMPG2), which is known to beinvolved in tumor growth, which also correlates with protein expressiondata. The data demonstrate that spatial protein expression can identifyimmune cell infiltration in ovarian cancer FFPE tissues sections andmoreover the spatial protein expression correlates with annotations by apathologist and with gene expression data which demonstrates the utilityof the methods described herein in identifying immune cells within atumor microenvironment. The methods described herein can also be used asa diagnostic tool and/or screening method. Additionally, spatial geneexpression correlating to different ovarian carcinomas within a tissuesection are distinguishable by the methods described herein.

Example 6. Spatial Proteomics and Spatial Gene Expression in FFPE CancerTissue Sections

Experiments were undertaken to determine whether analyte capture agentscould provide for spatial protein and gene expression analysis in FFPEcancer tissue sections. The tissue sections were prepared and analysiswas performed by the methods described in Example 4.

Lung Cancer

As shown in FIGS. 27A-D spatial gene and spatial protein expressionanalysis in a FFPE lung cancer tissue section. FIG. 27A shows an H&Estained lung cancer FFPE tissue section and FIGS. 27B and 27C, showspatial gene expression clustering (FIG. 27B) and spatial proteinexpression clustering (FIG. 27C), respectively. FIG. 27D shows a HLA-DRprotein spatial UMI plot. As described herein, HLA-DR is a marker of Tcell activation and FIG. 27D

The data demonstrate that spatial gene and spatial protein expressioncorrelate with each other which demonstrates the utility of the methodsdescribed herein in identifying spatial gene and protein expression inlung cancer FFPE tissue sections which can be used as a diagnostic tool.

Melanoma

As shown in FIGS. 28A-D spatial gene and spatial protein expressionanalysis in a FFPE melanoma tissue section. FIG. 28A shows an H&Estained lung cancer FFPE tissue section and FIGS. 28B and 28C, showspatial gene expression clustering (FIG. 28B) and spatial proteinexpression clustering (FIG. 28C), respectively. FIG. 28D shows HLA-DRprotein spatial UMI plot. As described herein, HLA-DR is a marker of Tcell activation and FIG. 28D

The data demonstrate that spatial gene and spatial protein expressioncorrelate with each other which demonstrates the utility of the methodsdescribed herein in identifying spatial gene and protein expression inmelanoma FFPE tissue sections which can be used as a diagnostic tool.

Other Tissues Assayed

In addition to the various FFPE tissue sections assayed as describedherein, other tissue types were assayed including: healthy brain tissue,breast cancer invasive lobular carcinoma tissue, healthy breast tissue,colon cancer tissue, healthy colon tissue, glioblastoma tissue, hearttissue, healthy lung tissue, prostate cancer tissue, healthy spleentissue, testes tissue, inflamed tonsil tissue, cervix tissue, and lymphnode tissue (data not shown).

Example 7. Spatial Protein Expression Correlates with ImmunofluorescenceStaining

FIGS. 29A-C show Vimentin (VIM) antibody immunofluorescence staining(FIG. 29A) and DAPI staining in a grade II invasive ductal carcinomaFFPE breast cancer tissue section. FIG. 29B shows spatial proteinexpression superimposed on the fluorescent image in FIG. 29A. FIG. 29Cshows spatial protein expression with a VIM specific analyte captureagent in the same tissue section. The data demonstrate a similar spatialdistribution between Vimentin antibody immunofluorescent staining (FIG.29A) and spatial protein expression with a VIM specific analyte captureagent (FIG. 29C) and demonstrates the utility of the methods describedherein to recapitulate spatial protein expression with analyte captureagents relative to immunofluorescent antibody staining.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. (canceled)
 2. A composition comprising: a) a spatial arraycomprising: (i) a first plurality of capture probes wherein the firstplurality of capture probes comprise a spatial barcode and a firstplurality of capture domains; and (ii) a second plurality of captureprobes wherein the second plurality of capture probes comprise a spatialbarcode and a second plurality of capture domains hybridized to aplurality of oligonucleotides from an analyte capture agent, wherein theanalyte capture agent comprises an analyte-binding moiety comprising anantibody or an antigen-binding fragment thereof, and wherein theoligonucleotides comprise an analyte capture sequence and an analytebinding moiety barcode; and b) a plurality of first probes, wherein afirst probe of the plurality of first probes is hybridized to a targetnucleic acid and a plurality of second probes, wherein a second probe ofthe plurality of second probes is hybridized to the target nucleic acidand wherein the second probe has a sequence complementary to the firstplurality of capture domains.
 3. The composition of claim 2, wherein thefirst plurality of capture probes further comprise one or morefunctional domains, a cleavage domain, a unique molecular identifier,and combinations thereof.
 4. The composition of claim 2, wherein thesecond plurality of capture probes further comprise one or morefunctional domains, a cleavage domain, a unique molecular identifier,and combinations thereof.
 5. The composition of claim 2, wherein thefirst plurality of capture domains comprise homopolymeric sequences andthe second plurality of capture domains comprise homopolymericsequences.
 6. The composition of claim 2, wherein the first plurality ofcapture domains are homopolymeric sequences and the second plurality ofcapture domains are non-homopolymeric sequences.
 7. The composition ofclaim 2, wherein the first plurality of capture domains arenon-homopolymeric sequences and the second plurality of capture domainsare non-homopolymeric sequences.
 8. The composition of claim 5, whereinthe first plurality of capture domains and the second plurality ofcapture domains are poly(T) sequences.
 9. The composition of claim 7,wherein the non-homopolymeric sequences of the first plurality ofcapture domains and the non-homopolymeric sequences of the secondplurality of capture domains are different.
 10. The composition of claim6, wherein the homopolymeric sequences of the first plurality of capturedomains are poly(T) sequences and the non-homopolymeric sequences of thesecond plurality of capture domains comprise a fixed sequence.
 11. Thecomposition of claim 10, wherein the fixed sequence comprises at leastone sequence selected from SEQ ID NO: 1 through SEQ ID NO:
 11. 12. Thecomposition of claim 2, wherein the spatial array further comprises oneor more protein dilution series.
 13. The composition of claim 2, whereinthe spatial array comprises a substrate comprising a plurality offeatures, wherein a feature of the plurality of features comprises abead or a well.
 14. The composition of claim 13, wherein the firstplurality of capture probes and the second plurality of capture probesare directly attached to the substrate.
 15. The composition of claim 13,wherein the first plurality of capture probes and the second pluralityof capture probes are indirectly attached to the substrate.
 16. Thecomposition of claim 2, wherein the first probe hybridized to the targetnucleic acid and the second probe hybridized to the target nucleic acidare ligated.
 17. The composition of claim 2, wherein the target nucleicacid is DNA.
 18. The composition of claim 2, wherein the target nucleicacid is RNA.
 19. The composition of claim 16, further comprising aRNase.
 20. The composition of claim 2, further comprising apermeabilization reagent.
 21. The composition of claim 20, wherein thepermeabilization reagent comprises a protease and a detergent.